biomapping

Epitope Tags in Protein ResearchTag Selection amp Immunotechniques

Choosing an Epitope Tag 3Purification 3Detection 5Cleavage sites 8High Throughput Expression 8Summary 9References 9

Epitope Tags 10Cellulose Binding Domain (CBD) 10Chloramphenicol Acetyl Transferase (CAT) 10Dihydrofolate Reductase (DHFR) 11FLAGreg 11Glutathione S-Transferase (GST) 13Green Fluorescent Protein (GFP) 14Hemagglutinin A (HA) 14Histidine (His) 15Herpes Simplex Virus (HSV) 16Luciferase 16Maltose-Binding Protein (MBP) 17c-Myc 17Protein A and Protein G 18Streptavidin 18T7 20Thioredoxin 21V5 21Vesicular Stomatitis Virus Glycoprotein (VSV-G) 22Yeast 2-hybrid tags B42 GAL4 LexA VP16 22Further reading 23

Protocols 24Protein Extraction 24

Sonication (E coli) 24Reagents 24Procedure 25

Freeze-thawing (cultured cells) 26Reagents 26Procedure 26

Detergent extraction (S cerevisiae) 27Reagents 27Procedure 27

Protein Purification 28His-tagged protein purification 28

Reagents 28Procedure 29Reagent compatibility chart 30

Immunoprecipitation 31Reagents 32

Direct Immunoprecipitation (microcolumn) 33Large scale (5 mL) procedure 34Indirect Immunoprecipitation 35

lsquoResin Firstrsquo method 36lsquoLysate Firstrsquo method 37Preparation for SDS-PAGE 38

Immunoblotting (Western Blotting) 38Reagents 39Protein Transfer 39Direct Detection 40Indirect Detection 41

Immunohistochemistry 42Reagents and Equipment 42Paraffin RemovalRehydration 43Antigen Retrieval (optional step for weak signal) 44

Enzymatic Method 44Microwave Retrieval 44

Inactivation of Peroxidase (if HRP detection used) 45Primary Antibody Reaction 46Secondary Reaction 47

Option 1 BiotinStreptavidin Detection 47Option 2 Enzyme-labeled Secondary Antibody 48

Development 49Counterstaining 50

Immunofluorescence 51Reagents and Equipment 52Cell Preparation 53Fixation 53

Methanol-Acetone Fixation 53Paraformaldehyde-TRITOnreg Fixation 53Paraformaldehyde-Methanol Fixation 54PEM-Ethanol Fixation 54

Application of Primary Antibody 55Application of Secondary Antibody 55Evaluation 56

ELISA 56Indirect ELISA 56

Reagents and Equipment 56Antigen Coating 57Primary Antibody Reaction 58Development 59

References 59

Contents

biomapping

Order 800-325-3010 Technical Service 800-325-5832 3

Choosing an Epitope TagUnlike DnA and RnA which can be targeted with complementary oligonucleotides protein detection and purification usually relies upon specific antibodies Although many antibodies are available commercially they do not cover all proteins especially if the protein has novel or unknown sequences

Raising polyclonal antibodies is time-consuming and expensive raising monoclonal antibodies even more so Epitope tags (also known as fusion tags or affinity tags) offer a convenient solution to this problem by acting as universal epitopes for detection and purification without disturbing the structure of the protein to which they are fused

One of the earliest tags was poly-arginine a run of five arginine residues created a basic region that allowed purification with cation exchange resin (Smith et al 1984) Since then the number of tags has steadily grown and so have the number of factors that can be considered when designing an expression study This guide lays out the features of different epitope tags to help with this design one of the main choices is whether the aim of expression is detection or purification of the protein

PurificationAll tags offer some means of purification but the purity convenience and cost of these platforms determine their suitability The best known tag is poly-histidine which is a form of Immobilized Metal Affinity Chromatography (IMAC) It is based upon the affinity of nickel for four or more consecutive histidine residues It is popular because of its ability to purify under denaturing conditions ease of reuse and reasonable price

-OOCNi

-OOC

-OOC

NCHN

CH2

N Resin

N

CH 2

N

ProteinCoordinated Bond

Nickel(II) ion

Figure 1 Binding of Histidine repeats to immobilized nickel ion via coordinated bonds

biomapping

Other tags with cost-effective purification resins are Maltose-Binding Protein (MBP) Cellulose-Binding Domain (CBD) and Glutathione S-Transferase (GST) These tags are approximately 40 times the size of metal affinity tags so they may interfere with the structure or function of the fusion partner However this can be beneficial tags such as MBP and GST can improve solubility (Dyson et al 2004) which is especially important when expressing proteins at high levels in prokaryotes as they have more primitive post-translational folding and processing machinery than eukaryotes

Tags that use antibody-based purification formats (e g FLAGreg HA HSV c-Myc) offer higher levels of purity but at a cost antibody resins are expensive to produce and cannot be reused easily However the high-purity often means that they are cost-effective in the long run as secondary purification steps can be avoided This is of particular use in high-throughput screening or where proteins are expressed at low levels e g while expression conditions are being optimized

Epitope tags can also be used for immobilization or attachment e g to investigate protein interactions If a purification resin is loaded with protein then it can be used as lsquobaitrsquo to capture binding partners (immunoprecipitation) In this case the specificity of the resin becomes crucial to avoid interference from contaminants

Biotin-based tags bind very tightly (Ka = 10-14 M) to streptavidin (Bayer et al 1990) so they are ideal for immobilizing proteins for studies where they will be subjected to repeated washes (e g ELISA and array-based assays)

DetectionThe ideal tag for detection would be large and hydrophilic with a strong antibody recognition site posi-tioned in an exposed region of the protein However large tags can interfere with protein structure and exposed regions are often functionally active so choosing a tag is not trivial

Because detection is performed under aqueous conditions a hydrophilic tag is more likely to be presented at the protein surface and thus accessible to antibodies and capture agents All tags are hydrophilic but some more than others Possession of charged (acid or basic) residues increases hydrophilicity and is a feature of naturally-ocurring epitopes

The hydrophilic nature of tags can also increase the solubility of the expression protein size and posi-tioning of the tag also contributes to this effect (Dyson et al 2004)

Large tags are often easier to detect because they are sterically more accessible to antibodies Even in the presence of SDS smaller tags can sometimes be folded inside the host protein and evade detection Using a tag that incorporates charged residues helps overcome this their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition

The FLAGreg epitope is a practical example of how size and charge phenomena can be exploited it is only eight amino acids long but all residues are charged (DYKDDDDK) so detection is very sensitive This sensitivity is enhanced 10-fold by tripling the size of the epitope to the lsquo3xFLAGtradersquo tandem repeat (DYKDHDGDYKDHDIDYKDDDK)

Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis However n-terminal placement seems to have a stronger effect on protein solubility possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al 2004)

Detection need not be restricted to immunodetection Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays respectively GFP can also be imaged in live cells so processes can be studied in real time rather than extrapolated from fixed sections A comparison of different detection methods is shown in the figures below

Comparison of Immunodetection Methods

Immunoblotting

Horseradishperoxidase (HRP)antibody conjugate

Substrate

Oxidized substrate+ signal (light or color change)

Epitope tagProtein

Blotting membrane

Immunocytochemistry

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Primary antibody

Immunofluorescence

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Fluorophore conjugatedto secondary antibody

Primary antibody

Signal viewed byfluoresencemicroscopy

ELISA (Enzyme-Linked ImmunoSorbent Assay)

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag

Protein in solution(eg cell lysate)

Primary antibody

Reaction vessel (eg 96-well plate)

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

Choosing an Epitope Tag 3Purification 3Detection 5Cleavage sites 8High Throughput Expression 8Summary 9References 9

Epitope Tags 10Cellulose Binding Domain (CBD) 10Chloramphenicol Acetyl Transferase (CAT) 10Dihydrofolate Reductase (DHFR) 11FLAGreg 11Glutathione S-Transferase (GST) 13Green Fluorescent Protein (GFP) 14Hemagglutinin A (HA) 14Histidine (His) 15Herpes Simplex Virus (HSV) 16Luciferase 16Maltose-Binding Protein (MBP) 17c-Myc 17Protein A and Protein G 18Streptavidin 18T7 20Thioredoxin 21V5 21Vesicular Stomatitis Virus Glycoprotein (VSV-G) 22Yeast 2-hybrid tags B42 GAL4 LexA VP16 22Further reading 23

Protocols 24Protein Extraction 24

Sonication (E coli) 24Reagents 24Procedure 25

Freeze-thawing (cultured cells) 26Reagents 26Procedure 26

Detergent extraction (S cerevisiae) 27Reagents 27Procedure 27

Protein Purification 28His-tagged protein purification 28

Reagents 28Procedure 29Reagent compatibility chart 30

Immunoprecipitation 31Reagents 32

Direct Immunoprecipitation (microcolumn) 33Large scale (5 mL) procedure 34Indirect Immunoprecipitation 35

lsquoResin Firstrsquo method 36lsquoLysate Firstrsquo method 37Preparation for SDS-PAGE 38

Immunoblotting (Western Blotting) 38Reagents 39Protein Transfer 39Direct Detection 40Indirect Detection 41

Immunohistochemistry 42Reagents and Equipment 42Paraffin RemovalRehydration 43Antigen Retrieval (optional step for weak signal) 44

Enzymatic Method 44Microwave Retrieval 44

Inactivation of Peroxidase (if HRP detection used) 45Primary Antibody Reaction 46Secondary Reaction 47

Option 1 BiotinStreptavidin Detection 47Option 2 Enzyme-labeled Secondary Antibody 48

Development 49Counterstaining 50

Immunofluorescence 51Reagents and Equipment 52Cell Preparation 53Fixation 53

Methanol-Acetone Fixation 53Paraformaldehyde-TRITOnreg Fixation 53Paraformaldehyde-Methanol Fixation 54PEM-Ethanol Fixation 54

Application of Primary Antibody 55Application of Secondary Antibody 55Evaluation 56

ELISA 56Indirect ELISA 56

Reagents and Equipment 56Antigen Coating 57Primary Antibody Reaction 58Development 59

References 59

Contents

biomapping

Order 800-325-3010 Technical Service 800-325-5832 3

Choosing an Epitope TagUnlike DnA and RnA which can be targeted with complementary oligonucleotides protein detection and purification usually relies upon specific antibodies Although many antibodies are available commercially they do not cover all proteins especially if the protein has novel or unknown sequences

Raising polyclonal antibodies is time-consuming and expensive raising monoclonal antibodies even more so Epitope tags (also known as fusion tags or affinity tags) offer a convenient solution to this problem by acting as universal epitopes for detection and purification without disturbing the structure of the protein to which they are fused

One of the earliest tags was poly-arginine a run of five arginine residues created a basic region that allowed purification with cation exchange resin (Smith et al 1984) Since then the number of tags has steadily grown and so have the number of factors that can be considered when designing an expression study This guide lays out the features of different epitope tags to help with this design one of the main choices is whether the aim of expression is detection or purification of the protein

PurificationAll tags offer some means of purification but the purity convenience and cost of these platforms determine their suitability The best known tag is poly-histidine which is a form of Immobilized Metal Affinity Chromatography (IMAC) It is based upon the affinity of nickel for four or more consecutive histidine residues It is popular because of its ability to purify under denaturing conditions ease of reuse and reasonable price

-OOCNi

-OOC

-OOC

NCHN

CH2

N Resin

N

CH 2

N

ProteinCoordinated Bond

Nickel(II) ion

Figure 1 Binding of Histidine repeats to immobilized nickel ion via coordinated bonds

biomapping

Other tags with cost-effective purification resins are Maltose-Binding Protein (MBP) Cellulose-Binding Domain (CBD) and Glutathione S-Transferase (GST) These tags are approximately 40 times the size of metal affinity tags so they may interfere with the structure or function of the fusion partner However this can be beneficial tags such as MBP and GST can improve solubility (Dyson et al 2004) which is especially important when expressing proteins at high levels in prokaryotes as they have more primitive post-translational folding and processing machinery than eukaryotes

Tags that use antibody-based purification formats (e g FLAGreg HA HSV c-Myc) offer higher levels of purity but at a cost antibody resins are expensive to produce and cannot be reused easily However the high-purity often means that they are cost-effective in the long run as secondary purification steps can be avoided This is of particular use in high-throughput screening or where proteins are expressed at low levels e g while expression conditions are being optimized

Epitope tags can also be used for immobilization or attachment e g to investigate protein interactions If a purification resin is loaded with protein then it can be used as lsquobaitrsquo to capture binding partners (immunoprecipitation) In this case the specificity of the resin becomes crucial to avoid interference from contaminants

Biotin-based tags bind very tightly (Ka = 10-14 M) to streptavidin (Bayer et al 1990) so they are ideal for immobilizing proteins for studies where they will be subjected to repeated washes (e g ELISA and array-based assays)

DetectionThe ideal tag for detection would be large and hydrophilic with a strong antibody recognition site posi-tioned in an exposed region of the protein However large tags can interfere with protein structure and exposed regions are often functionally active so choosing a tag is not trivial

Because detection is performed under aqueous conditions a hydrophilic tag is more likely to be presented at the protein surface and thus accessible to antibodies and capture agents All tags are hydrophilic but some more than others Possession of charged (acid or basic) residues increases hydrophilicity and is a feature of naturally-ocurring epitopes

The hydrophilic nature of tags can also increase the solubility of the expression protein size and posi-tioning of the tag also contributes to this effect (Dyson et al 2004)

Large tags are often easier to detect because they are sterically more accessible to antibodies Even in the presence of SDS smaller tags can sometimes be folded inside the host protein and evade detection Using a tag that incorporates charged residues helps overcome this their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition

The FLAGreg epitope is a practical example of how size and charge phenomena can be exploited it is only eight amino acids long but all residues are charged (DYKDDDDK) so detection is very sensitive This sensitivity is enhanced 10-fold by tripling the size of the epitope to the lsquo3xFLAGtradersquo tandem repeat (DYKDHDGDYKDHDIDYKDDDK)

Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis However n-terminal placement seems to have a stronger effect on protein solubility possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al 2004)

Detection need not be restricted to immunodetection Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays respectively GFP can also be imaged in live cells so processes can be studied in real time rather than extrapolated from fixed sections A comparison of different detection methods is shown in the figures below

Comparison of Immunodetection Methods

Immunoblotting

Horseradishperoxidase (HRP)antibody conjugate

Substrate

Oxidized substrate+ signal (light or color change)

Epitope tagProtein

Blotting membrane

Immunocytochemistry

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Primary antibody

Immunofluorescence

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Fluorophore conjugatedto secondary antibody

Primary antibody

Signal viewed byfluoresencemicroscopy

ELISA (Enzyme-Linked ImmunoSorbent Assay)

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag

Protein in solution(eg cell lysate)

Primary antibody

Reaction vessel (eg 96-well plate)

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

Order 800-325-3010 Technical Service 800-325-5832 3

Choosing an Epitope TagUnlike DnA and RnA which can be targeted with complementary oligonucleotides protein detection and purification usually relies upon specific antibodies Although many antibodies are available commercially they do not cover all proteins especially if the protein has novel or unknown sequences

Raising polyclonal antibodies is time-consuming and expensive raising monoclonal antibodies even more so Epitope tags (also known as fusion tags or affinity tags) offer a convenient solution to this problem by acting as universal epitopes for detection and purification without disturbing the structure of the protein to which they are fused

One of the earliest tags was poly-arginine a run of five arginine residues created a basic region that allowed purification with cation exchange resin (Smith et al 1984) Since then the number of tags has steadily grown and so have the number of factors that can be considered when designing an expression study This guide lays out the features of different epitope tags to help with this design one of the main choices is whether the aim of expression is detection or purification of the protein

PurificationAll tags offer some means of purification but the purity convenience and cost of these platforms determine their suitability The best known tag is poly-histidine which is a form of Immobilized Metal Affinity Chromatography (IMAC) It is based upon the affinity of nickel for four or more consecutive histidine residues It is popular because of its ability to purify under denaturing conditions ease of reuse and reasonable price

-OOCNi

-OOC

-OOC

NCHN

CH2

N Resin

N

CH 2

N

ProteinCoordinated Bond

Nickel(II) ion

Figure 1 Binding of Histidine repeats to immobilized nickel ion via coordinated bonds

biomapping

Other tags with cost-effective purification resins are Maltose-Binding Protein (MBP) Cellulose-Binding Domain (CBD) and Glutathione S-Transferase (GST) These tags are approximately 40 times the size of metal affinity tags so they may interfere with the structure or function of the fusion partner However this can be beneficial tags such as MBP and GST can improve solubility (Dyson et al 2004) which is especially important when expressing proteins at high levels in prokaryotes as they have more primitive post-translational folding and processing machinery than eukaryotes

Tags that use antibody-based purification formats (e g FLAGreg HA HSV c-Myc) offer higher levels of purity but at a cost antibody resins are expensive to produce and cannot be reused easily However the high-purity often means that they are cost-effective in the long run as secondary purification steps can be avoided This is of particular use in high-throughput screening or where proteins are expressed at low levels e g while expression conditions are being optimized

Epitope tags can also be used for immobilization or attachment e g to investigate protein interactions If a purification resin is loaded with protein then it can be used as lsquobaitrsquo to capture binding partners (immunoprecipitation) In this case the specificity of the resin becomes crucial to avoid interference from contaminants

Biotin-based tags bind very tightly (Ka = 10-14 M) to streptavidin (Bayer et al 1990) so they are ideal for immobilizing proteins for studies where they will be subjected to repeated washes (e g ELISA and array-based assays)

DetectionThe ideal tag for detection would be large and hydrophilic with a strong antibody recognition site posi-tioned in an exposed region of the protein However large tags can interfere with protein structure and exposed regions are often functionally active so choosing a tag is not trivial

Because detection is performed under aqueous conditions a hydrophilic tag is more likely to be presented at the protein surface and thus accessible to antibodies and capture agents All tags are hydrophilic but some more than others Possession of charged (acid or basic) residues increases hydrophilicity and is a feature of naturally-ocurring epitopes

The hydrophilic nature of tags can also increase the solubility of the expression protein size and posi-tioning of the tag also contributes to this effect (Dyson et al 2004)

Large tags are often easier to detect because they are sterically more accessible to antibodies Even in the presence of SDS smaller tags can sometimes be folded inside the host protein and evade detection Using a tag that incorporates charged residues helps overcome this their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition

The FLAGreg epitope is a practical example of how size and charge phenomena can be exploited it is only eight amino acids long but all residues are charged (DYKDDDDK) so detection is very sensitive This sensitivity is enhanced 10-fold by tripling the size of the epitope to the lsquo3xFLAGtradersquo tandem repeat (DYKDHDGDYKDHDIDYKDDDK)

Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis However n-terminal placement seems to have a stronger effect on protein solubility possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al 2004)

Detection need not be restricted to immunodetection Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays respectively GFP can also be imaged in live cells so processes can be studied in real time rather than extrapolated from fixed sections A comparison of different detection methods is shown in the figures below

Comparison of Immunodetection Methods

Immunoblotting

Horseradishperoxidase (HRP)antibody conjugate

Substrate

Oxidized substrate+ signal (light or color change)

Epitope tagProtein

Blotting membrane

Immunocytochemistry

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Primary antibody

Immunofluorescence

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Fluorophore conjugatedto secondary antibody

Primary antibody

Signal viewed byfluoresencemicroscopy

ELISA (Enzyme-Linked ImmunoSorbent Assay)

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag

Protein in solution(eg cell lysate)

Primary antibody

Reaction vessel (eg 96-well plate)

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

biomapping

Other tags with cost-effective purification resins are Maltose-Binding Protein (MBP) Cellulose-Binding Domain (CBD) and Glutathione S-Transferase (GST) These tags are approximately 40 times the size of metal affinity tags so they may interfere with the structure or function of the fusion partner However this can be beneficial tags such as MBP and GST can improve solubility (Dyson et al 2004) which is especially important when expressing proteins at high levels in prokaryotes as they have more primitive post-translational folding and processing machinery than eukaryotes

Tags that use antibody-based purification formats (e g FLAGreg HA HSV c-Myc) offer higher levels of purity but at a cost antibody resins are expensive to produce and cannot be reused easily However the high-purity often means that they are cost-effective in the long run as secondary purification steps can be avoided This is of particular use in high-throughput screening or where proteins are expressed at low levels e g while expression conditions are being optimized

Epitope tags can also be used for immobilization or attachment e g to investigate protein interactions If a purification resin is loaded with protein then it can be used as lsquobaitrsquo to capture binding partners (immunoprecipitation) In this case the specificity of the resin becomes crucial to avoid interference from contaminants

Biotin-based tags bind very tightly (Ka = 10-14 M) to streptavidin (Bayer et al 1990) so they are ideal for immobilizing proteins for studies where they will be subjected to repeated washes (e g ELISA and array-based assays)

DetectionThe ideal tag for detection would be large and hydrophilic with a strong antibody recognition site posi-tioned in an exposed region of the protein However large tags can interfere with protein structure and exposed regions are often functionally active so choosing a tag is not trivial

Because detection is performed under aqueous conditions a hydrophilic tag is more likely to be presented at the protein surface and thus accessible to antibodies and capture agents All tags are hydrophilic but some more than others Possession of charged (acid or basic) residues increases hydrophilicity and is a feature of naturally-ocurring epitopes

The hydrophilic nature of tags can also increase the solubility of the expression protein size and posi-tioning of the tag also contributes to this effect (Dyson et al 2004)

Large tags are often easier to detect because they are sterically more accessible to antibodies Even in the presence of SDS smaller tags can sometimes be folded inside the host protein and evade detection Using a tag that incorporates charged residues helps overcome this their hydrophilicity drives them to the surface and provides a strong motif for antibody recognition

The FLAGreg epitope is a practical example of how size and charge phenomena can be exploited it is only eight amino acids long but all residues are charged (DYKDDDDK) so detection is very sensitive This sensitivity is enhanced 10-fold by tripling the size of the epitope to the lsquo3xFLAGtradersquo tandem repeat (DYKDHDGDYKDHDIDYKDDDK)

Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis However n-terminal placement seems to have a stronger effect on protein solubility possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al 2004)

Detection need not be restricted to immunodetection Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays respectively GFP can also be imaged in live cells so processes can be studied in real time rather than extrapolated from fixed sections A comparison of different detection methods is shown in the figures below

Comparison of Immunodetection Methods

Immunoblotting

Horseradishperoxidase (HRP)antibody conjugate

Substrate

Oxidized substrate+ signal (light or color change)

Epitope tagProtein

Blotting membrane

Immunocytochemistry

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Primary antibody

Immunofluorescence

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Fluorophore conjugatedto secondary antibody

Primary antibody

Signal viewed byfluoresencemicroscopy

ELISA (Enzyme-Linked ImmunoSorbent Assay)

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag

Protein in solution(eg cell lysate)

Primary antibody

Reaction vessel (eg 96-well plate)

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

Order 800-325-3010 Technical Service 800-325-5832 5

Tags can be placed anywhere within the protein but C-terminal tags are less likely to interfere with any signal peptides and act as an indicator of complete protein synthesis However n-terminal placement seems to have a stronger effect on protein solubility possibly because the tag has a chance to fold before the remainder of the fusion protein is translated and possibly misfolded (Dyson et al 2004)

Detection need not be restricted to immunodetection Chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) can both be measured by enzyme or fluorescence assays respectively GFP can also be imaged in live cells so processes can be studied in real time rather than extrapolated from fixed sections A comparison of different detection methods is shown in the figures below

Comparison of Immunodetection Methods

Immunoblotting

Horseradishperoxidase (HRP)antibody conjugate

Substrate

Oxidized substrate+ signal (light or color change)

Epitope tagProtein

Blotting membrane

Immunocytochemistry

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Primary antibody

Immunofluorescence

Epitope tag ProteinEpitope tag Protein

Tissue section fixed onto glass slide

Fluorophore conjugatedto secondary antibody

Primary antibody

Signal viewed byfluoresencemicroscopy

ELISA (Enzyme-Linked ImmunoSorbent Assay)

Enzyme conjugatedto secondaryantibody

Substrate

Coloredprecipitate

Epitope tag

Protein in solution(eg cell lysate)

Primary antibody

Reaction vessel (eg 96-well plate)

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

biomapping

Cleavage SitesAlthough small tags may not interfere with folding it is often better to work with the native proteins especially if attempting to determine the structure To this end there are a number of proteases and site combinations available for removal of tags These sites are often pre-engineered into expression vectors and in the case of enterokinase actually form part of the tag sequence

Proteases can be heat-inactivated post-cleavage or more commonly used as resin conjugates for efficient removal

Cleavage Site Enzyme

Asp-Asp-Asp-Asp-Lys-X Enterokinase

Ile-GluAsp-Gly-Arg-X Factor Xa

Leu-Val-Pro-Arg-X-Gly-Ser Thrombin

Glu-Asn-Leu-Tyr-Phe-Gln-X-Gly Tobacco Etch Virus (TEV) protease

Table 1 Proteolytic cleavage sites used for epitope tag removal

High-Throughput ExpressionBecause protein expression is still an inexact science even small expression projects can require hundreds of combinations given all of the factors that can be assessed Choice of epitope tag can influence how easy these experiments are to carry out both practically (e g are multiwell purification plate formats avail-able for screening) and scientifically (e g will it be possible to detect the protein at the expected levels of expression)

To this end it can be useful to incorporate more than one tag typically at opposite termini to avoid the risk of interference An example of this might be to use a sensitive lsquodetectionrsquo tag as well as a lsquopurificationrsquo tag so that expression can be optimized quickly using the detection tag yet purified with the purification tag without the need to reclone

For large expression projects it becomes impractical to test many combinations for each protein and this has led to studies to predict optimal parameters for successful expression One of the biggest hurdles (especially in prokaryotes) is maintaining protein solubility Many eukaryotic proteins require post-transla-tional modifications in order to fold correctly which coupled with the artificially high protein concentra-tions in the cell can lead to misfolding and aggregation Accumulation of misfolded protein is often toxic but even if the proteins are packaged in inclusion bodies refolding them is not trivial

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

Order 800-325-3010 Technical Service 800-325-5832 7

SummaryEpitope tags simplify protein expression studies by providing universal methods for purification and detection They can also aid soluble expression in prokaryotic systems and add biochemical functions to aid investigation Choice of tag depends upon many factors and involves a degree of compromise in most cases but one of the most important considerations is the downstream analysis of the protein

lsquoPurificationrsquo tags such as poly-histidine are relatively inexpensive to use but lack specificity and sensitivity when used for immunodetection

lsquoDetectionrsquo tags are ideally suited for immunodetection and offer a rapid (but more expensive) option for purification

One compromise solution is combinatorial tagging using a mixture of tags on a protein can give the best of both worlds and accelerate protein expression studies

ReferencesBayer EA Ben-Hur H Wilchek M (1990) ldquoIsolation and properties of streptavidin rdquoMethods Enzymol 184 80ndash9

Dyson MR Shadbolt SP Vincent KJ Perera RL McCafferty J (2004) ldquoProduction of soluble mammalian proteins in Escherichia coli identification of protein features that correlate with successful expression rdquo BMC Biotechnol Dec 14 4(1) 32

Hernan R Heuermann K Brizzard B (2000) ldquoMultiple epitope tagging of expressed proteins for enhanced detection rdquo Biotechniques Apr 8(4) 789ndash93

Smith JC Derbyshire RB Cook E Dunthorne L Viney J Brewer SJ Sassenfeld HM Bell LD (1984) ldquoChemical synthesis and cloning of a poly(arginine)-coding gene fragment designed to aid polypeptide purification rdquo Gene Dec 32(3) 321ndash7

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

biomapping

Epitope Tags The biochemical properties of the popular epitope tags are summarized along with the tools available for their study

Chloramphenicol Acetyl Transferase (CAT) This 24 kDa tag is also used as a reporter gene and retains its functionality when fused to most proteins This means that it can be used to measure levels of expression directly without the need for PAGE or immunodetection Purification is achieved using chloramphenicol-Sepharosereg

Cat No Anti-Chloramphenicol Acetyl Transferase (CAT) antibody produced in rabbit

C9336- 5ML

Dihydrofolate Reductase (DHFR) This is a well-characterized 25 kDa protein involved in the thymidine biosynthesis pathway Purification of tagged proteins can be achieved by methotrexate-linked resin

Cat No Anti-DHFR C-terminal antibody produced in rabbit

D0942-100UGAnti-DHFR n-terminal antibody produced in rabbit

D1067-100UG

Legend

Detection Purification Controls

Order 800-325-3010 Technical Service 800-325-5832 9

FLAGreg

The octapeptide sequence is highly charged (DYKDDDDK) and useful for sensitive detection especially if concatenated as the 3xFLAGtrade epitope (DYKDHDGDYKDHDIDYKDDDK) This facilitates the study of low-abundance proteins and the optimization of difficult protein expression projects

It is also popular as a purification tag the high level of purification can eliminate the need for further clean-up The FLAG sequence also contains an enterokinase cleavage site to allow removal if the tag is placed at the n-terminus

There are four types of FLAG antibodiesM1 exhibits Calcium-dependent binding but does not recognize Met-FLAG

fusions or unprocessed cytoplasmically-expressed proteins

M2 recognizes all types of fusions

Polyclonal ANTI-FLAGreg recognizes all types of fusions

M5 Only recognizes n-terminal Met-FLAG fusions and is not recommended for use in E coli expression systems

Cat No Monoclonal ANTI-FLAG M1 mouseF3040- 2MG F3040-1MG F3040-5MG Monoclonal ANTI-FLAG M2 mouse purified immunoglobulinF3165- 2MGF3165-1MGF3165-5MGMonoclonal ANTI-FLAGreg M2 mouse Affinity Purified IgGF1804-200UG F1804-1MG F1804-5MG Monoclonal ANTI-FLAG M2-Peroxidase (HRP) antibody produced in mouseA8592- 2MG A8592-1MG A8592-531MG

biomapping

Cat No Monoclonal ANTI-FLAG M2-Alkaline Phosphatase antibody produced in mouseA9469- 2MG A9469-1MG A9469-531MG Monoclonal ANTI-FLAG M2-Cytrade3 antibody produced in mouseA9594- 2MG A9594-1MG A9594-531MG Monoclonal ANTI-FLAG M2-Biotin antibody produced in mouseF9291- 2MG F9291-1MG F9291-531MG Monoclonal ANTI-FLAG M2-FITC Conjugate antibody produced in mouseF4049- 2MGF4049-1MGF4049-531MG Polyclonal ANTI-FLAG antibody produced in rabbit F7425- 2MG Monoclonal ANTI-FLAG M5 antibody produced in mouseF4042- 2MG F4042-1MG F4042-5MG Monoclonal ANTI-FLAG M5-Biotin antibody produced in mouseF2922- 2MGANTI-FLAG M2 Affinity GelA2220-1MLA2220-5MLA2220-10MLA2220-25MLEZviewtrade Red ANTI-FLAG M2 Affinity GelF2426-1MLF2426-531ML

Order 800-325-3010 Technical Service 800-325-5832 11

Cat No ANTI-FLAG M1 Agarose Affinity GelA4596-1MLA4596-5MLA4596-10MLA4596-25MLFLAGreg Immunoprecipitation KitFLAG IPT1-1KTANTI-FLAGreg High Sensitivity M2 coated 96-well platesP2983-1EAFLAG PeptideF3290-4MGF3290-25MG3x FLAGtrade PeptideF4799-4MGF4799-25MGAmino-terminal Met-FLAG-BAPtrade Fusion ProteinP5975- 1MGCarboxy-terminal FLAG-BAP Fusion ProteinP7457- 1MGAmino-terminal FLAG-BAP Fusion ProteinP7582- 1MG

Glutathione S-Transferase (GST)GST was one of the first epitope tags to be used it can be placed on the n or C-terminus and can enhance the solubility of expressed proteins Purification is achieved with glutathione-conjugated resin

Cat No Anti-GST peroxidase conjugate produced in rabbitA7340- 5MLMonoclonal Anti-GST antibody produced in mouseG1160- 2MLG1160- 5ML

biomapping

Cat No Glutathione AgaroseG4510-1MLG4510-5MLG4510-10MLG4510-50MLPre-packed columns (3 3 2 5 mL)G3907-1SETEZviewtrade Red Glutathione Affinity ResinE6406-1MLE6406-5X1ML

Green Fluorescent Protein (GFP)Unique among epitope tags GFP is an autofluorescent 27 kDa tag that can be directly detected in living cells by fluorescent microscopy

Cat NoMonoclonal Anti-Green Fluorescent Protein (GFP) antibody produced in mouse

G6539- 2ML

G6539- 5ML

Monoclonal Anti-GFP n-terminal antibody produced in mouse

G6795-200UG

Anti-GFP n-terminal antibody produced in rabbit

G1544-100UG

Hemagglutinin A (HA)Derived from the binding domain of the Influenza hemagglutinin protein HA contains a high proportion of charged residues (YPYDVPDYA) so it is likely to form a strong antibody recognition site

Cat NoMonoclonal Anti-HA antibody produced in mouse

H3663-200UL

Monoclonal Anti-HA Peroxidase antibody produced in mouse

H6533-1VL (vial of 0 5 mL)

Order 800-325-3010 Technical Service 800-325-5832 13

Cat NoMonoclonal Anti-HA Alkaline Phosphatase antibody produced in mouse

A5477-500UG

Monoclonal Anti-HA FITC antibody produced in mouse

H7411-100UG

Monoclonal Anti-HA TRITC antibody produced in mouse

H9037-200UG

Monoclonal Anti-HA-Biotin antibody produced in mouse

B9183-100UG

Monoclonal Anti-HA Agarose antibody produced in mouse

A2095-1ML

EZviewtrade Red Anti-HA Affinity GelE6779-1ML

E6779-531ML

Anti-HA Immunoprecipitation KitIP0010-1KT

Histidine (His) This is by far the most widely used purification tag it allows purification with economical nickel affinity resins These resins tolerate denaturing conditions (useful for purifying solubilized proteins from inclusion bodies) and can be reused Binding specificity is less than that of antibody-based resins so a second purification step is often necessary this also removes the high imidazole concentration used for elution Acidic elution has been used as a low-salt alternative to imidazole and cobalt can been used in place of nickel to increase specificity Metalloproteins and histidine-rich proteins (e g Chloramphenicol Acetyltransferase) also bind to these resins so it is advisible to use suitable controls

Cat NoMonoclonal Anti-polyhistidine Alkaline Phosphatase antibody produced in mouse

A5588- 5ML

Monoclonal Anti-polyhistidine Peroxidase antibody produced in mouse

A7058-1VL (vial of 0 5 mL)

Monoclonal Anti-polyhistidine antibody produced in mouse

H1029- 2ML

H1029- 5ML

biomapping

Cat NoHIS-Selectreg Nickel Affinity GelP6611-5ML

P6611-25ML

P6611-100ML

P6611-500ML

HIS-Select Cobalt Affinity Gel H8162-5ML

H8162-25ML

H8162-100ML

HIS-Select HF Nickel Affinity GelH0537-10ML

H0537-25ML

H0537-100ML

H0537-500ML

EZviewtrade Red HIS-Select HC Nickel Affinity GelE3528-1ML

E3528-531ML

HIS-Select Spin Columns H7787-10EA

H7787-50EA

HIS-Selectreg High Capacity (HC) Nickel Coated PlatesS5563-1EA

HIS-Select High Sensitivity (HS) Nickel Coated PlatesS5688-1EA

HIS-Select Wash amp Elution Buffer KitH5288-500ML

H5413-250ML

Order 800-325-3010 Technical Service 800-325-5832 15

Herpes Simplex Virus (HSV)HSV is derived from the glycoprotein D precursor envelope protein and is short (QPELAPEDPED) so it is unlikely to interfere with protein structure or function

Cat NoAnti-HSV antibody produced in rabbit

H6030-200UG

Anti-HSV Peroxidase antibody produced in rabbit

H0912-200UL

HSV Peptide

H4640-4MG

H4640-25MG

LuciferaseThis is better known as a reporter gene for expression assays Antibodies allow results to be verified by immunological methods

Cat NoMonoclonal Anti-Luciferase antibody produced in mouseL2164- 2ML

Maltose-Binding Protein (MBP) The size (43 kDa) of this tag contributes to its ability to increase soluble protein production in E coli Along with GST and Thioredoxin it is popular as a lsquosolubilityrsquo tag Purification is achieved using low-cost amylose resin an Asn10 spacer between the tag and the protein improving resin binding

Cat NoMonoclonal Anti-Maltose Binding Protein antibody produced in mouse

M6295- 2ML

M6295- 5ML

Monoclonal Anti-Maltose Binding Protein Alkaline Phosphatase antibody produced in mouse

A3963- 5ML

Monoclonal Anti-Maltose Binding Protein Peroxidase antibody produced in mouse

A4213-1VL (vial of 0 5 mL)

biomapping

c-Mycc-Myc (or myc) has been extensively used for Western blotting immunoprecipitation and flow cytometry Its small size (EQKLISEEDL) means that it is unlikely to perturb (or enhance) protein folding and it has been used at both n and C-termini

Cat NoMonoclonal Anti-c-Myc antibody produced in mouse

M4439-100UL

Anti-c-Myc antibody produced in rabbit

C3956- 2MG

Anti-c-Myc Peroxidase antibody produced in rabbit

A5598-500UG

Monoclonal Anti-c-Myc Biotin antibody produced in mouse

B7554-100UG

Monoclonal Anti-c-Myc FITC antibody produced in mouse

F2047-100UG

Anti-c-Myc Agarose Affinity Gel antibody produced in rabbitA7470-1ML

EZviewtrade Red Anti-c-Myc Affinity GelE6654-1ML

E6654-531ML

Anti-c-Myc Immunoprecipitation KitIP0020-1KT

c-Myc Peptide

M2435-4MGM2435-25MG

Protein A and Protein G These bacterial lsquosuperantigensrsquo bind to all forms of IgG They can be fused to proteins for purification using IgG but they are most often employed as conjugates for generic secondary detection or purification of antibodies

Cat NoProtein A AgaroseP2545-1ML

P2545-5ML

Order 800-325-3010 Technical Service 800-325-5832 17

Cat NoEZviewtrade Red Protein A Affinity GelP6486-1ML

P6486-5X1ML

EZview Red Protein G Affinity GelE3403-1ML

E3403-5X1ML

Protein G Immunoprecipitation KitIP50-1KT

StreptavidinBiotin The affinity of the streptavidin-biotin interaction (10-14 M) means that it can withstand harsh conditions so it is popular as a method of immobilizing proteins (e g in production of protein arrays) Streptavidin can be incorporated as a full-length truncated or mutated protein and biotin can be incorporated by fusing a protein domain that becomes biotinylated in vivo (e g biotin-carboxy carrier protein)

Cat NoAnti-Streptavidin antibody produced in rabbit

S6390-1ML

Monoclonal Anti-Biotin antibody produced in mouse

B7653- 2ML

B7653- 5ML

Anti-Biotin antibody produced in goat

B3640-1MG

StreptavidinminusPeroxidase from Streptomyces avidiniiS5512-250UG

S5512- 5MG

S5512-2MG

Anti-BiotinminusPeroxidase antibody produced in goat

A4541- 25ML

A4541- 5ML

A4541-1ML

Monoclonal Anti-BiotinminusPeroxidase antibody produced in mouse

A0185-1VL (vial of 0 5 mL)

StreptavidinminusPeroxidase Polymer Ultrasensitive

S2438-250UG

biomapping

Cat NoStreptavidinminusAlkaline Phosphatase from Streptomyces avidiniiS2890-250UG

S2890-1MG

Anti-BiotinminusAlkaline Phosphatase antibody produced in goat

A7064- 25ML

A7064- 5ML

A7064-1ML

Monoclonal Anti-BiotinndashAlkaline Phosphatase antibody produced in mouse

A6561- 2ML

A6561- 5ML

Monoclonal Anti-BiotinndashCytrade3 antibody produced in mouse

C5585- 2ML

C5585- 5ML

StreptavidinminusCy3 from Streptomyces avidiniiS6402-1ML

StreptavidinminusGold from Streptomyces avidiniiS9059- 4ML

S9059-2ML

StreptavidinminusFITC from Streptomyces avidiniiS3762- 1MG

S3762- 5MG

S3762-1MG

Anti-BiotinminusFITC antibody produced in goat

F6762- 5ML

Monoclonal Anti-BiotinndashFITC antibody produced in mouse

F4024- 2ML

F4024- 5ML

Biotin-4-Fluorescein

B9431-5MG

StreptavidinminusFluorescent Polymer Ultrasensitive

S2313-250UG

Streptavidin-R-Phycoerythrin from Streptomyces avidinii

S3402-1ML

Order 800-325-3010 Technical Service 800-325-5832 19

Cat NoStreptavidinminusAgarose from Streptomyces avidinii S1638-1ML

S1638-5ML

Streptavidin immobilized on Agarose CL-4B85881-1ML

85881-5ML

EZviewtrade Red Streptavidin Affinity GelE5529-1ML

E5529-531ML

BiotinminusAgaroseB0519-5ML

B0519-25ML

StreptavidinminusIron Oxide Particles from Streptomyces avidinii S2415-10ML

Streptavidin from Strepotmyces avidinii recombinant

S0677-1MG

S0677-5MG

S0677-25MG

Biotin powder ge99

B4639-1G

B4639-5G

More tools for StreptavidinBiotin are available Search for lsquoStreptavidinrsquo or lsquoBiotinrsquo on our website at sigma-aldrichcom

T7 This is a 260-residue tag derived from the gene 10 product of T7 phage may enhance expression levels in E coli

Cat NoMonoclonal Anti-T7 tag antibody produced in mouse

T8823-200UG

Monoclonal Anti-T7 tag Peroxidase conjugate antibody produced in mouse

T3699-1VL

biomapping

Thioredoxin Despite its small size (11 kDa) thioredoxin is very effective as a lsquosolubilizingrsquo tag (As with all solubilizing tags solubility does not necessarily mean functional folding and fusion proteins can precipitate when their tag is cleaved) Thioredoxin is unique among epitope tags in being stable up to 80 degC and can confer some thermostability onto its fusion partner This way cellular proteins can be heat-denatured without affecting the fusion protein Purification can either be achieved using phenylarsine oxide resin or antibody-conjugated resin

Cat NoAnti-Thioredoxin antibody produced in rabbitT0803- 2MLT0803- 5MLAnti-ThioredoxinndashAgarose antibody produced in rabbit A2582-1MLThioredoxin from Escherichia coli T0910-1MG

V5A small tag (GKPIPnPLLGLDST) derived from residues 95-108 of the PV proteins of the Paramyxovirus SV5 Vectors purification resins and antibodies are widely available

Cat NoMonoclonal Anti-V5-Peroxidase antibody produced in mouseV2260-1VL (vial of 0 5 mL)Anti-V5 antibody produced in rabbitV8137- 2MGMonoclonal Anti-V5 antibody produced in mouseV8012-50UGMonoclonal Anti-V5-Cytrade3 antibody produced in mouseV4014-100UGAnti-V5 Agarose Affinity Gel antibody produced in mouseA7345-1MLV5 PeptideV7754-4MG

Order 800-325-3010 Technical Service 800-325-5832 21

Vesicular Stomatitis Virus Glycoprotein (VSV-G) The VSV-G antibody recognizes the five C-terminal residues of the tag (YTDIEMnRLGK) Like many tags it is found as a cassette in commercially available vectors to aid cloning

Cat NoMonoclonal Anti-VSV-G Peroxidase antibody produced in mouseA5977-1VLAnti-VSV-G antibody produced in rabbitV4888-200UGMonoclonal Anti-VSV-Glycoprotein Agarose antibody produced in mouseA1970-1MLVSV-G PeptideV7887-4MGV7887-25MG

Yeast 2-hybrid tags B42 GAL4 LexA VP16These tags are used to detect protein interactions in the yeast 2-hybrid system acting as DnA binding (GAL4 LexA) domains or transcriptional activators (B42 GAL4 VP16) Antibodies against these tags allow results to be validated and investigated further

Cat NoAnti-B42 antibody produced in rabbitB9808-200UGAnti-GAL4 Activation domain antibody produced in rabbitG9293-200UGAnti-GAL4 DnA-BD antibody produced in rabbitG3042-200UGAnti-Lex A antibody produced in rabbitL0415-100UGAnti-VP16 antibody produced in rabbitV4388-200UL

biomapping

Further ReadingEinhauer A Jungbauer A J (2001) ldquoThe FLAG peptide a versatile fusion tag for the purification of recombinant proteins rdquo Biochem Biophys Methods Oct 30 49 (1ndash3) 455ndash465

Makrides SC (1996) ldquoStrategies for achieving high-level expression of genes in Eschericia coli rdquo Microbiol Rev Sep 60 (3) 512ndash538

Sheibani n (1999) ldquoProkaryotic gene fusion expression systems and their use in structural and functional studies of proteins rdquo Prep Biochem Biotechnol Feb 29 (1) 77ndash90

Stevens RC (2000) ldquoDesign of high-throughput methods of protein production for structural biology rdquo RC Structure Sep 15 8 (9) R177ndash85

Terpe K (2003) ldquoOverview of tag protein fusions from molecular and biochemical fundamentals to commercial systems rdquo Appl Microbiol Biotechnol Jan 60 (5) 523ndash533

Waugh DS (2005) ldquoMaking the most of affinity tags rdquo Trends Biotechnol Jun 23 (6) 316ndash20

Order 800-325-3010 Technical Service 800-325-5832 23

ProtocolsThis section outlines some of the commonly used protocols in protein extraction purification and detection

Protein Extraction In order to purify proteins they must be released from the cell and solubilized Unfortunately this also releases proteases so inhibitors must be added to limit proteolysis Three popular methods for protein extraction are sonication freeze-thawing and detergent extraction All three can be used with any type of starting material but the most common applications are used here as examples

Sonication (Escherichia coli)Sonication is achieved by sending high frequency sound waves through a cell suspension The shear forces induced by the sound waves disrupt solid matter breaking down tissues and cells Large masses of cells can be processed but the procedure is fairly hands-on so it is best suited to small numbers of medium or large scale preparations

Reagents

Sonication bufferTris-HCl pH 7 5 50 mM naCl 50ndash200 mM Reducing agent 5ndash10 mM

Note Most reducing agents are incompatible with nickel purification resins

Protease Inhibitor Cocktails

Cat NoGeneral Use lyophilized powderP2714-1BTLBacterial lyophilized powderP8465-5MLP8465-25MLEDTA-Free solution in DMSOP8849-1MLP8849-5ML

To view more protease and phosphatase inhibitor cocktails visit sigmacomprotinhib

Order 800-325-3010 Technical Service 800-325-5832 23

ProtocolsThis section outlines some of the commonly used protocols in protein extraction purification and detection

Protein Extraction In order to purify proteins they must be released from the cell and solubilized Unfortunately this also releases proteases so inhibitors must be added to limit proteolysis Three popular methods for protein extraction are sonication freeze-thawing and detergent extraction All three can be used with any type of starting material but the most common applications are used here as examples

Sonication (Escherichia coli)Sonication is achieved by sending high frequency sound waves through a cell suspension The shear forces induced by the sound waves disrupt solid matter breaking down tissues and cells Large masses of cells can be processed but the procedure is fairly hands-on so it is best suited to small numbers of medium or large scale preparations

Reagents

Sonication bufferTris-HCl pH 7 5 50 mM naCl 50ndash200 mM Reducing agent 5ndash10 mM

Note Most reducing agents are incompatible with nickel purification resins

Protease Inhibitor Cocktails

Cat NoGeneral Use lyophilized powderP2714-1BTLBacterial lyophilized powderP8465-5MLP8465-25MLEDTA-Free solution in DMSOP8849-1MLP8849-5ML

To view more protease and phosphatase inhibitor cocktails visit sigmacomprotinhib

biomapping

Procedure

1 Harvest cells by centrifugation (3000 3 g for 15 minutes)

2 Resuspend in 1ndash4 volumes (massvolume) of ice-cold sonication buffer

3 Incubate on ice for 10 minutes Add protease inhibitor cocktail and mix thoroughly Note Some protease inhibitors (e g PMSF) have short half-lives in aqueous solution so it is important to add them fresh

4 Keeping the cell suspension on ice sonicate in 10 second bursts allowing 20 second cooling period in between Ten repeats should be sufficient Note Try to minimize foaming as this can denature protein Sonicator horn can shatter glass if it comes into direct contact with it

5 Pellet cell debris by centrifugation (20000 3 g for 20 minutes) Collect and retain soluble fraction Note If the protein is expressed in inclusion bodies then the insoluble fraction should be retained and solubilized in a denaturing buffer e g denaturing equilibration buffer for histidine purification described later

4˚C

Order 800-325-3010 Technical Service 800-325-5832 25

Freeze-thawing (cultured cells)Freeze-thawing uses the growth of ice crystals to puncture and break apart cells It is ideal for studying low-abundance proteins due to the high concentration of sample First published by Rudolph et al in 1999 (Anal Biochem 269 66ndash71)

Reagents

Lysis bufferTris-HCl pH 7 8 20 mMKCl 600 mMGlycerol 20

Procedure

1 Aspirate medium and wash in ice-cold PBS

2 Scrape cells in a minimal volume of ice-cold PBS and transfer to a microcentrifuge tube

3 Pellet cells by centrifugation (10000 3 g for 30 seconds) and carefully discard the supernatant

4 Resuspend in lysis buffer (50 μL per 10 cm plate)

5 Flash freeze in liquid nitrogen

biomapping

6 Thaw on ice and repeat twice

7 Add 250 units of Benzonasereg (Cat No E1013) and incubate at room temperature for 10 minutes Note Benzonase reduces the viscosity of the lysate by digesting genomic DNA making the sample easier to pipette and pass over chromatography media

Detergent Extraction (S cerevisiae)Detergent extraction is suitable for all scales of extraction and circumvents the need for enzymic lysis when used with Saccharomyces cerevisiae All steps are performed at 4 degC

Reagents

Cat NoCelLytictrade Y Cell Lysis ReagentC4482-50MLC4482-500MLYeast Protease Inhibitor Cocktail DMSO solutionP8215-5MLP8215-25ML

To view our full line of lysis reagents visit sigmacomlysis

Procedure

1 Harvest cells by centrifugation (3000 3 g for 5 minutes) andresuspend in lysis buffer (2 5ndash5 mL per gram of cell paste) Note Addition of 5ndash10 mM DTT will increase protein yield especially if cells were harvested after log phase

2 Incubate with gentle shaking for 15ndash30 minutes

4˚C

Order 800-325-3010 Technical Service 800-325-5832 25

Freeze-thawing (cultured cells)Freeze-thawing uses the growth of ice crystals to puncture and break apart cells It is ideal for studying low-abundance proteins due to the high concentration of sample First published by Rudolph et al in 1999 (Anal Biochem 269 66ndash71)

Reagents

Lysis bufferTris-HCl pH 7 8 20 mMKCl 600 mMGlycerol 20

Procedure

1 Aspirate medium and wash in ice-cold PBS

2 Scrape cells in a minimal volume of ice-cold PBS and transfer to a microcentrifuge tube

3 Pellet cells by centrifugation (10000 3 g for 30 seconds) and carefully discard the supernatant

4 Resuspend in lysis buffer (50 μL per 10 cm plate)

5 Flash freeze in liquid nitrogen

biomapping

6 Thaw on ice and repeat twice

7 Add 250 units of Benzonasereg (Cat No E1013) and incubate at room temperature for 10 minutes Note Benzonase reduces the viscosity of the lysate by digesting genomic DNA making the sample easier to pipette and pass over chromatography media

Detergent Extraction (S cerevisiae)Detergent extraction is suitable for all scales of extraction and circumvents the need for enzymic lysis when used with Saccharomyces cerevisiae All steps are performed at 4 degC

Reagents

Cat NoCelLytictrade Y Cell Lysis ReagentC4482-50MLC4482-500MLYeast Protease Inhibitor Cocktail DMSO solutionP8215-5MLP8215-25ML

To view our full line of lysis reagents visit sigmacomlysis

Procedure

1 Harvest cells by centrifugation (3000 3 g for 5 minutes) andresuspend in lysis buffer (2 5ndash5 mL per gram of cell paste) Note Addition of 5ndash10 mM DTT will increase protein yield especially if cells were harvested after log phase

2 Incubate with gentle shaking for 15ndash30 minutes

4˚C

biomapping

6 Thaw on ice and repeat twice

7 Add 250 units of Benzonasereg (Cat No E1013) and incubate at room temperature for 10 minutes Note Benzonase reduces the viscosity of the lysate by digesting genomic DNA making the sample easier to pipette and pass over chromatography media

Detergent Extraction (S cerevisiae)Detergent extraction is suitable for all scales of extraction and circumvents the need for enzymic lysis when used with Saccharomyces cerevisiae All steps are performed at 4 degC

Reagents

Cat NoCelLytictrade Y Cell Lysis ReagentC4482-50MLC4482-500MLYeast Protease Inhibitor Cocktail DMSO solutionP8215-5MLP8215-25ML

To view our full line of lysis reagents visit sigmacomlysis

Procedure

1 Harvest cells by centrifugation (3000 3 g for 5 minutes) andresuspend in lysis buffer (2 5ndash5 mL per gram of cell paste) Note Addition of 5ndash10 mM DTT will increase protein yield especially if cells were harvested after log phase

2 Incubate with gentle shaking for 15ndash30 minutes

4˚C

Order 800-325-3010 Technical Service 800-325-5832 27

3 Pellet cell debris by centrifugation (12000 3 g for 20 minutes) and retain supernatant

Protein PurificationTraditionally proteins were purified by chromatography based on their native properties (e g size charge and hydrophobicity) however epitope tags allow any protein to be purified by affinity chromatography which simplifies the procedure and can yield pure preparations after one round of purification

The two most popular types of affinity purification are those employing metal affinity resins (for histidine tags) and antibody-resin conjugates (often termed immunoprecipitation)

Histidine-tagged Protein PurificationHistidine-tagged proteins are purified using the affinity of histidine repeats for nickel ions (Figure 1) This protocol is for small-scale preparations using HIS-Selectreg Spin Columns (Cat No H7787) but large scale preparations can be performed using affinity gel (Cat No P6611)

Histidine tags can sometimes be hidden by protein folding preventing binding to the resin Because nickel resin is compatible with denaturing agents this can sometimes be solved by purifying under denaturing conditions simply solubilize the protein in 8 M urea (Cat No U1250) during extraction and use the denaturing buffer recipes Bear in mind that it is difficult to functionally refold denatured proteins

Reagents

Equilibration Buffer Native DenaturingSodium phosphate pH 8 0 50 mM 50 mMSodium chloride 0 3 M mdashUrea mdash 8 MWash BufferSodium phosphate pH 8 0 50 mM 50 mMImidazole 5 mM 5 mMSodium chloride 0 3 M mdashUrea mdash 8 MElution BufferSodium phosphate pH 8 0 50 mM 50 mMImidazole 250 mM 250 mMSodium chloride 0 3 M mdashUrea mdash 8 M

biomapping

Note Any buffers containing urea must be made fresh daily The elution buffer pH may need to be varied between 4 5ndash6 0 (depending on the protein) because some recombinant proteins with histi-dine tags will not elute in the pH 5 0 to 6 0 range If the tagged recombinant proteins will not elute in this range try a pH as low as 4 5

Note The equilibration and washbuffers should be supplemented with 1ndash10 mM imidazole and 0 15ndash0 5 M sodium chloride to reduce non-specific protein binding Due to the unique selectivity of the chelate 5 mM imidazole in the wash buffer is sufficient to obtain high purity samples Consult the reagent compatibility chart for the use of other reagents

Procedure

1 Place HIS-Selectreg Spin Column in a collection tube

2 Add 600 μL of equilibration buffer to the spin column

3 Close lid onto spin column and centrifuge at 1000 rpm (82 3 g) at room temperature for 1 minute Note HIS-Select Spin Columns may also be used with an appropriate vacuum manifold in place of the centrifugation step

4 Remove spin column from collection tube

5 Empty the collection tube and replace the spin column

6 Centrifuge prepared cell extract through the column 600 μL at a time (82 3 g for 1 minute)

7 Remove spin column from collection tube Remove and retain the flow-through for later analysis (if required) Note Presence of expressed protein could indicate a problem with binding to the resin

8 Using a new collection tube wash unbound protein from the spin column with 600 μL of wash buffer (82 3 g for1 minute) Remove and retain flow through for analysis (if required) Note Presence of expressed protein could indicate problems with resin binding or wash buffer conditions

9 Repeat wash step with 600 μL of wash buffer

10 Using new collection tube elute the target protein using up to 500 μL of elution buffer (82 3 g for 1 minute)

Order 800-325-3010 Technical Service 800-325-5832 29

biomapping

Reagent Compatibility Chart

Compound Max conc CommentsImidazole histidine 10 mM Compete with histidine for nickel binding

High concentrations reduce yieldChelating agents (e g EDTA EGTA) none Can remove nickel ions from the resinGlycerol 50 Helps stabilize proteins2-mercaptoethanol 20 mM Only use in extraction buffer

Can reduce nickel ionsOther reducing agents none not recommendednonionic detergents (e g TWEEnreg) 2Glycine none Binds to nickel use histidine or Imidazole instead

Tips

bullnon-specific binding can be reduced by increasing the imidazole concentration in the wash buffer

bullImmunoblotting the crude lysate can be used to verify protein expression

bullUse denaturing conditions if you suspect the tag is buried (e g if there is poor recovery despite strong expression)

bullGlycerol salt detergents and reducing agents can help reduce protein precipitation

bullRetain all fractions for PAGE analysis it makes problems easier to diagnose and solve

Immunoprecipitation

Immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting is routinely used in a variety of applications

bullStudying proteinprotein interactionsbullDetermining the molecular masses of protein antigensbullMonitoring post-translational modifications

It also enables concentration and detection of rare proteins which otherwise would be difficult to detect The technique is essentially small-scale affinity purification protein is purified on an agarose-linked antibody complex Agarose is insoluble so all of the antibody-binding partners can be separated from solution by centrifugation

Many agarose conjugates are available commercially (especially those linked to antibodies against epitope tags) but superantigen (Protein A G or L) conjugates are also available which allow indirect immunoprecipitation if an antibody against the target protein is added first The choice of superantigen conjugate depends on the species origin and isotype of the primary antibody (Table 2 see page 31)

Order 800-325-3010 Technical Service 800-325-5832 31

Affinity Spectra of Superantigens

Species Immunoglobulin Protein A Protein G Protein LHuman IgG (normal) ++++ ++++ ++++

IgG1 ++++ ++++ ++++

IgG2 ++++ ++++ ++++

IgG3 - ++++ ++++

IgG4 ++++ ++++ ++++

IgM - - ++++

IgA - - ++++

IgE - - ++++

IgD - - ++++

Fab ++ ++ ++++

K light chains - - ++++

L light chains - - -

ScFv ++ - ++++

Mouse IgG1 + ++++ ++++

IgG2a ++++ ++++ ++++

IgG2b +++ +++ ++++

IgG3 ++ +++ ++++

Rat IgG1 - + ++++

IgG2a - ++++ ++++

IgG2b - ++ ++++

IgG2c + ++ ++++

Bovine IgG ++ ++++ -

Cat IgG ++++ - nA

Chicken IgG - + ++

Dog IgG ++++ ++++ +

Goat IgG +- ++ -

Guinea Pig IgG ++++ ++ ++

Hamster IgG + ++ ++++

Horse IgG ++ ++++ +-

Pig IgG +++ +++ ++++

Rabbit IgG ++++ +++ +

Sheep IgG +- ++ -

Table 2

biomapping

ReagentsAntibody-agarose conjugate or primary antibody and superantigen conjugate e g anti-c Myc agarose (Cat No A7470) and protein G agarose (Cat No E3403)

Note Perform all steps using microcentrifuge tubes on ice unless noted otherwise

Direct Immunoprecipitation (microcolumn)This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin

1 Pipette 40ndash100 microL of the 50 suspension of resin into a microcentrifuge tube or spin-column

2 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

3 Wash the resin 5 times with 1 mL of PBS using centrifugation to pellet the resin

4 Add clarified bacterial lysate or cell extract to the resin pellet

5 Bring the volume to at least 200 microL with PBS or RIPA buffer if needed

HNTG bufferHEPES pH 7 5 20 mMnaCl 150 mMTRITOnreg X-100 0 1 (wv)Glycerol 10 (wv)

Washing buffer (Ice cold)HnTG bufferor PBS pH 7 4 (Cat no P4417)or other buffers e g RIPA buffer

6 Centrifuge prepared cell extract through the column 600 μL at a time (82 3 g for 1 minute)

7 Remove spin column from collection tube Remove and retain the flow-through for later analysis (if required) Note Presence of expressed protein could indicate a problem with binding to the resin

8 Using a new collection tube wash unbound protein from the spin column with 600 μL of wash buffer (82 3 g for1 minute) Remove and retain flow through for analysis (if required) Note Presence of expressed protein could indicate problems with resin binding or wash buffer conditions

9 Repeat wash step with 600 μL of wash buffer

10 Using new collection tube elute the target protein using up to 500 μL of elution buffer (82 3 g for 1 minute)

Order 800-325-3010 Technical Service 800-325-5832 29

biomapping

Reagent Compatibility Chart

Compound Max conc CommentsImidazole histidine 10 mM Compete with histidine for nickel binding

High concentrations reduce yieldChelating agents (e g EDTA EGTA) none Can remove nickel ions from the resinGlycerol 50 Helps stabilize proteins2-mercaptoethanol 20 mM Only use in extraction buffer

Can reduce nickel ionsOther reducing agents none not recommendednonionic detergents (e g TWEEnreg) 2Glycine none Binds to nickel use histidine or Imidazole instead

Tips

bullnon-specific binding can be reduced by increasing the imidazole concentration in the wash buffer

bullImmunoblotting the crude lysate can be used to verify protein expression

bullUse denaturing conditions if you suspect the tag is buried (e g if there is poor recovery despite strong expression)

bullGlycerol salt detergents and reducing agents can help reduce protein precipitation

bullRetain all fractions for PAGE analysis it makes problems easier to diagnose and solve

Immunoprecipitation

Immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting is routinely used in a variety of applications

bullStudying proteinprotein interactionsbullDetermining the molecular masses of protein antigensbullMonitoring post-translational modifications

It also enables concentration and detection of rare proteins which otherwise would be difficult to detect The technique is essentially small-scale affinity purification protein is purified on an agarose-linked antibody complex Agarose is insoluble so all of the antibody-binding partners can be separated from solution by centrifugation

Many agarose conjugates are available commercially (especially those linked to antibodies against epitope tags) but superantigen (Protein A G or L) conjugates are also available which allow indirect immunoprecipitation if an antibody against the target protein is added first The choice of superantigen conjugate depends on the species origin and isotype of the primary antibody (Table 2 see page 31)

Order 800-325-3010 Technical Service 800-325-5832 31

Affinity Spectra of Superantigens

Species Immunoglobulin Protein A Protein G Protein LHuman IgG (normal) ++++ ++++ ++++

IgG1 ++++ ++++ ++++

IgG2 ++++ ++++ ++++

IgG3 - ++++ ++++

IgG4 ++++ ++++ ++++

IgM - - ++++

IgA - - ++++

IgE - - ++++

IgD - - ++++

Fab ++ ++ ++++

K light chains - - ++++

L light chains - - -

ScFv ++ - ++++

Mouse IgG1 + ++++ ++++

IgG2a ++++ ++++ ++++

IgG2b +++ +++ ++++

IgG3 ++ +++ ++++

Rat IgG1 - + ++++

IgG2a - ++++ ++++

IgG2b - ++ ++++

IgG2c + ++ ++++

Bovine IgG ++ ++++ -

Cat IgG ++++ - nA

Chicken IgG - + ++

Dog IgG ++++ ++++ +

Goat IgG +- ++ -

Guinea Pig IgG ++++ ++ ++

Hamster IgG + ++ ++++

Horse IgG ++ ++++ +-

Pig IgG +++ +++ ++++

Rabbit IgG ++++ +++ +

Sheep IgG +- ++ -

Table 2

biomapping

ReagentsAntibody-agarose conjugate or primary antibody and superantigen conjugate e g anti-c Myc agarose (Cat No A7470) and protein G agarose (Cat No E3403)

Note Perform all steps using microcentrifuge tubes on ice unless noted otherwise

Direct Immunoprecipitation (microcolumn)This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin

1 Pipette 40ndash100 microL of the 50 suspension of resin into a microcentrifuge tube or spin-column

2 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

3 Wash the resin 5 times with 1 mL of PBS using centrifugation to pellet the resin

4 Add clarified bacterial lysate or cell extract to the resin pellet

5 Bring the volume to at least 200 microL with PBS or RIPA buffer if needed

HNTG bufferHEPES pH 7 5 20 mMnaCl 150 mMTRITOnreg X-100 0 1 (wv)Glycerol 10 (wv)

Washing buffer (Ice cold)HnTG bufferor PBS pH 7 4 (Cat no P4417)or other buffers e g RIPA buffer

biomapping

Reagent Compatibility Chart

Compound Max conc CommentsImidazole histidine 10 mM Compete with histidine for nickel binding

High concentrations reduce yieldChelating agents (e g EDTA EGTA) none Can remove nickel ions from the resinGlycerol 50 Helps stabilize proteins2-mercaptoethanol 20 mM Only use in extraction buffer

Can reduce nickel ionsOther reducing agents none not recommendednonionic detergents (e g TWEEnreg) 2Glycine none Binds to nickel use histidine or Imidazole instead

Tips

bullnon-specific binding can be reduced by increasing the imidazole concentration in the wash buffer

bullImmunoblotting the crude lysate can be used to verify protein expression

bullUse denaturing conditions if you suspect the tag is buried (e g if there is poor recovery despite strong expression)

bullGlycerol salt detergents and reducing agents can help reduce protein precipitation

bullRetain all fractions for PAGE analysis it makes problems easier to diagnose and solve

Immunoprecipitation

Immunoprecipitation (IP) followed by SDS-PAGE and immunoblotting is routinely used in a variety of applications

bullStudying proteinprotein interactionsbullDetermining the molecular masses of protein antigensbullMonitoring post-translational modifications

It also enables concentration and detection of rare proteins which otherwise would be difficult to detect The technique is essentially small-scale affinity purification protein is purified on an agarose-linked antibody complex Agarose is insoluble so all of the antibody-binding partners can be separated from solution by centrifugation

Many agarose conjugates are available commercially (especially those linked to antibodies against epitope tags) but superantigen (Protein A G or L) conjugates are also available which allow indirect immunoprecipitation if an antibody against the target protein is added first The choice of superantigen conjugate depends on the species origin and isotype of the primary antibody (Table 2 see page 31)

Order 800-325-3010 Technical Service 800-325-5832 31

Affinity Spectra of Superantigens

Species Immunoglobulin Protein A Protein G Protein LHuman IgG (normal) ++++ ++++ ++++

IgG1 ++++ ++++ ++++

IgG2 ++++ ++++ ++++

IgG3 - ++++ ++++

IgG4 ++++ ++++ ++++

IgM - - ++++

IgA - - ++++

IgE - - ++++

IgD - - ++++

Fab ++ ++ ++++

K light chains - - ++++

L light chains - - -

ScFv ++ - ++++

Mouse IgG1 + ++++ ++++

IgG2a ++++ ++++ ++++

IgG2b +++ +++ ++++

IgG3 ++ +++ ++++

Rat IgG1 - + ++++

IgG2a - ++++ ++++

IgG2b - ++ ++++

IgG2c + ++ ++++

Bovine IgG ++ ++++ -

Cat IgG ++++ - nA

Chicken IgG - + ++

Dog IgG ++++ ++++ +

Goat IgG +- ++ -

Guinea Pig IgG ++++ ++ ++

Hamster IgG + ++ ++++

Horse IgG ++ ++++ +-

Pig IgG +++ +++ ++++

Rabbit IgG ++++ +++ +

Sheep IgG +- ++ -

Table 2

biomapping

ReagentsAntibody-agarose conjugate or primary antibody and superantigen conjugate e g anti-c Myc agarose (Cat No A7470) and protein G agarose (Cat No E3403)

Note Perform all steps using microcentrifuge tubes on ice unless noted otherwise

Direct Immunoprecipitation (microcolumn)This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin

1 Pipette 40ndash100 microL of the 50 suspension of resin into a microcentrifuge tube or spin-column

2 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

3 Wash the resin 5 times with 1 mL of PBS using centrifugation to pellet the resin

4 Add clarified bacterial lysate or cell extract to the resin pellet

5 Bring the volume to at least 200 microL with PBS or RIPA buffer if needed

HNTG bufferHEPES pH 7 5 20 mMnaCl 150 mMTRITOnreg X-100 0 1 (wv)Glycerol 10 (wv)

Washing buffer (Ice cold)HnTG bufferor PBS pH 7 4 (Cat no P4417)or other buffers e g RIPA buffer

Order 800-325-3010 Technical Service 800-325-5832 31

Affinity Spectra of Superantigens

Species Immunoglobulin Protein A Protein G Protein LHuman IgG (normal) ++++ ++++ ++++

IgG1 ++++ ++++ ++++

IgG2 ++++ ++++ ++++

IgG3 - ++++ ++++

IgG4 ++++ ++++ ++++

IgM - - ++++

IgA - - ++++

IgE - - ++++

IgD - - ++++

Fab ++ ++ ++++

K light chains - - ++++

L light chains - - -

ScFv ++ - ++++

Mouse IgG1 + ++++ ++++

IgG2a ++++ ++++ ++++

IgG2b +++ +++ ++++

IgG3 ++ +++ ++++

Rat IgG1 - + ++++

IgG2a - ++++ ++++

IgG2b - ++ ++++

IgG2c + ++ ++++

Bovine IgG ++ ++++ -

Cat IgG ++++ - nA

Chicken IgG - + ++

Dog IgG ++++ ++++ +

Goat IgG +- ++ -

Guinea Pig IgG ++++ ++ ++

Hamster IgG + ++ ++++

Horse IgG ++ ++++ +-

Pig IgG +++ +++ ++++

Rabbit IgG ++++ +++ +

Sheep IgG +- ++ -

Table 2

biomapping

ReagentsAntibody-agarose conjugate or primary antibody and superantigen conjugate e g anti-c Myc agarose (Cat No A7470) and protein G agarose (Cat No E3403)

Note Perform all steps using microcentrifuge tubes on ice unless noted otherwise

Direct Immunoprecipitation (microcolumn)This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin

1 Pipette 40ndash100 microL of the 50 suspension of resin into a microcentrifuge tube or spin-column

2 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

3 Wash the resin 5 times with 1 mL of PBS using centrifugation to pellet the resin

4 Add clarified bacterial lysate or cell extract to the resin pellet

5 Bring the volume to at least 200 microL with PBS or RIPA buffer if needed

HNTG bufferHEPES pH 7 5 20 mMnaCl 150 mMTRITOnreg X-100 0 1 (wv)Glycerol 10 (wv)

Washing buffer (Ice cold)HnTG bufferor PBS pH 7 4 (Cat no P4417)or other buffers e g RIPA buffer

biomapping

ReagentsAntibody-agarose conjugate or primary antibody and superantigen conjugate e g anti-c Myc agarose (Cat No A7470) and protein G agarose (Cat No E3403)

Note Perform all steps using microcentrifuge tubes on ice unless noted otherwise

Direct Immunoprecipitation (microcolumn)This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) Antibody interactions are often sensitive to changes in buffer composition (less so with superantigens) so for best results we recommend adhering to the protocol supplied with the antibody resin

1 Pipette 40ndash100 microL of the 50 suspension of resin into a microcentrifuge tube or spin-column

2 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

3 Wash the resin 5 times with 1 mL of PBS using centrifugation to pellet the resin

4 Add clarified bacterial lysate or cell extract to the resin pellet

5 Bring the volume to at least 200 microL with PBS or RIPA buffer if needed

HNTG bufferHEPES pH 7 5 20 mMnaCl 150 mMTRITOnreg X-100 0 1 (wv)Glycerol 10 (wv)

Washing buffer (Ice cold)HnTG bufferor PBS pH 7 4 (Cat no P4417)or other buffers e g RIPA buffer

6 Incubate for 1 5 hours on a rotator at room temperature or at 4 degC

7 Wash the resin 4 times with 1 mL of PBS or lysis buffer

8 After the final wash aspirate the supernatant and leave ~10 microL above the beads

9 Centrifuge at full speed for 10 seconds in a microcentrifuge Discard liquid

Large scale (5 mL) procedureImmunoprecipitation can also be used for affinity purification on larger scales This procedure is taken from the protocol of anti-c-Myc agarose (Cat No A7470) but can be adapted for other resins

1 Place an empty chromatography column e g 10 mL gravity column (Cat No 54806) on a firm support and rinse with PBS

2 Allow the buffer to drain from the column leaving residual PBS in the column to aid in packing the resin

3 Thoroughly suspend the vial of resin to make a uniform suspension and immediately transfer the desired volume to the column

Order 800-325-3010 Technical Service 800-325-5832 33

4 Allow the agarose bed to settle Do not let it dry out

5 Wash with three sequential 5 mL aliquots of 0 1 M ammonium hydroxide pH 11ndash12 followed by three sequential 5 mL aliquots of PBS

6 Load the lysate on the column under gravity flow Note Depending upon the fusion protein and the flow rate not all of the protein may bind Multiple passes over the column or closing the loaded column and incubating it on a rotator for about 1 hour may improve the binding efficiency

7 Collect the flow through of unbound protein for analysis

8 Wash the column with PBS until the OD280 of the flow through is lt0 01

9 Elute the bound c-Myc tagged fusion protein from the column with 10 x 1 mL aliquots of 0 1 M ammonium hydroxide at pH 11ndash12 into vials containing 30ndash50 microL of 1 n acetic acid for neutralization Note The column may lose activity after prolonged exposure to low pH

biomapping

Indirect ImmunoprecipitationIndirect immunoprecipitation allows a wider range of proteins to be purified superantigen resins are used to lsquoprecipitatersquo the antibody complexes so all that is required is an antibody against the target protein Because there are two binding reactions (superantigen resin-antibody and antibody-lysate) there are two variations of this technique resin first or lysate first

1 Wash agarose conjugate twice with washing buffer centrifuge for 10 seconds at 12000 3 g at room temperature Discard supernatant Note If agarose conjugate is a powder reconstitute it with deionized water and allow it to swell for 5 minutes

2 Resuspend agarose conjugate in washing buffer (50 suspension)

lsquoResin Firstrsquo method

3 Divide agarose conjugate into aliquots of 50ndash100 microL (25ndash50 microL agarosebed volume) in microcentrifuge tubes

4 Add to each tube 10 microL of primary antibody at appropriate dilution (refer to the antibody specifications)

5 Incubate for 15ndash60 minutes at room temperature gently mixing the sample on rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

Order 800-325-3010 Technical Service 800-325-5832 35

7 Wash samples at least 3 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

8 Add to each tube 0 1ndash1 0 mL of cell lysate

9 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

10 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

11 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

lsquoLysate Firstrsquo method

1 Wash agarose conjugate twice with washing buffer centrifuge for 10 seconds at 12000 3 g at room temperature Discard supernatant Note If agarose conjugate is a powder reconstitute it with deionized water and allow it to swell for 5 minutes

2 Resuspend agarose conjugate in washing buffer (50 suspension)

biomapping

3 Take cell lysate sample (0 1ndash1 0 mL) and add 10 microL of antibody at appropriate dilution (refer to product specification)

4 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

5 Add 50ndash100 microL of agarose conjugate suspension (25ndash50 microL agarosebed volume) Incubate for 15ndash60 minutes at 4 degC gently mixing the sample with a rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

7 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discard the supernatant

Order 800-325-3010 Technical Service 800-325-5832 37

Preparation for SDS-PAGE

1 Resuspend each pellet in 25ndash100 microL of Laemmli sample buffer to a final concentration of 13 sample buffer Heat samples at 95 degC for 5 minutes

2 Centrifuge for 30 seconds at 12000 3 g at room temperature

3 Collect supernatant Samples can be loaded straight away or stored in sample buffer at ndash70 degC

Immunoblotting (Western Blotting)Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting It catalyzes the oxidation of luminol or TMB (33prime55prime-Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) Chemiluminescent detection is many times more sensitive than colorimetric but is more expensive and laborious Colorimetric detection can be performed on the bench and allows signal development to be observed directly

Immunodetection can be performed directly or indirectly Direct detection employs HRP-conjugated antibodies to the protein or tag whereas indirect is performed in two stages using an unlabeled lsquoprimaryrsquo antibody against the protein or tag and a labeled lsquosecondaryrsquo antibody against the first Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat No A7058 and Cat No H1029) and will give results for a wide spectrum of other antibodies However optimal results are achieved if the protocol specific to the antibody is followed

biomapping

Indirect ImmunoprecipitationIndirect immunoprecipitation allows a wider range of proteins to be purified superantigen resins are used to lsquoprecipitatersquo the antibody complexes so all that is required is an antibody against the target protein Because there are two binding reactions (superantigen resin-antibody and antibody-lysate) there are two variations of this technique resin first or lysate first

1 Wash agarose conjugate twice with washing buffer centrifuge for 10 seconds at 12000 3 g at room temperature Discard supernatant Note If agarose conjugate is a powder reconstitute it with deionized water and allow it to swell for 5 minutes

2 Resuspend agarose conjugate in washing buffer (50 suspension)

lsquoResin Firstrsquo method

3 Divide agarose conjugate into aliquots of 50ndash100 microL (25ndash50 microL agarosebed volume) in microcentrifuge tubes

4 Add to each tube 10 microL of primary antibody at appropriate dilution (refer to the antibody specifications)

5 Incubate for 15ndash60 minutes at room temperature gently mixing the sample on rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

Order 800-325-3010 Technical Service 800-325-5832 35

7 Wash samples at least 3 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

8 Add to each tube 0 1ndash1 0 mL of cell lysate

9 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

10 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

11 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

lsquoLysate Firstrsquo method

1 Wash agarose conjugate twice with washing buffer centrifuge for 10 seconds at 12000 3 g at room temperature Discard supernatant Note If agarose conjugate is a powder reconstitute it with deionized water and allow it to swell for 5 minutes

2 Resuspend agarose conjugate in washing buffer (50 suspension)

biomapping

3 Take cell lysate sample (0 1ndash1 0 mL) and add 10 microL of antibody at appropriate dilution (refer to product specification)

4 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

5 Add 50ndash100 microL of agarose conjugate suspension (25ndash50 microL agarosebed volume) Incubate for 15ndash60 minutes at 4 degC gently mixing the sample with a rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

7 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discard the supernatant

Order 800-325-3010 Technical Service 800-325-5832 37

Preparation for SDS-PAGE

1 Resuspend each pellet in 25ndash100 microL of Laemmli sample buffer to a final concentration of 13 sample buffer Heat samples at 95 degC for 5 minutes

2 Centrifuge for 30 seconds at 12000 3 g at room temperature

3 Collect supernatant Samples can be loaded straight away or stored in sample buffer at ndash70 degC

Immunoblotting (Western Blotting)Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting It catalyzes the oxidation of luminol or TMB (33prime55prime-Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) Chemiluminescent detection is many times more sensitive than colorimetric but is more expensive and laborious Colorimetric detection can be performed on the bench and allows signal development to be observed directly

Immunodetection can be performed directly or indirectly Direct detection employs HRP-conjugated antibodies to the protein or tag whereas indirect is performed in two stages using an unlabeled lsquoprimaryrsquo antibody against the protein or tag and a labeled lsquosecondaryrsquo antibody against the first Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat No A7058 and Cat No H1029) and will give results for a wide spectrum of other antibodies However optimal results are achieved if the protocol specific to the antibody is followed

biomapping

7 Wash samples at least 3 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

8 Add to each tube 0 1ndash1 0 mL of cell lysate

9 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

10 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

11 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discarding supernatant

lsquoLysate Firstrsquo method

1 Wash agarose conjugate twice with washing buffer centrifuge for 10 seconds at 12000 3 g at room temperature Discard supernatant Note If agarose conjugate is a powder reconstitute it with deionized water and allow it to swell for 5 minutes

2 Resuspend agarose conjugate in washing buffer (50 suspension)

biomapping

3 Take cell lysate sample (0 1ndash1 0 mL) and add 10 microL of antibody at appropriate dilution (refer to product specification)

4 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

5 Add 50ndash100 microL of agarose conjugate suspension (25ndash50 microL agarosebed volume) Incubate for 15ndash60 minutes at 4 degC gently mixing the sample with a rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

7 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discard the supernatant

Order 800-325-3010 Technical Service 800-325-5832 37

Preparation for SDS-PAGE

1 Resuspend each pellet in 25ndash100 microL of Laemmli sample buffer to a final concentration of 13 sample buffer Heat samples at 95 degC for 5 minutes

2 Centrifuge for 30 seconds at 12000 3 g at room temperature

3 Collect supernatant Samples can be loaded straight away or stored in sample buffer at ndash70 degC

Immunoblotting (Western Blotting)Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting It catalyzes the oxidation of luminol or TMB (33prime55prime-Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) Chemiluminescent detection is many times more sensitive than colorimetric but is more expensive and laborious Colorimetric detection can be performed on the bench and allows signal development to be observed directly

Immunodetection can be performed directly or indirectly Direct detection employs HRP-conjugated antibodies to the protein or tag whereas indirect is performed in two stages using an unlabeled lsquoprimaryrsquo antibody against the protein or tag and a labeled lsquosecondaryrsquo antibody against the first Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat No A7058 and Cat No H1029) and will give results for a wide spectrum of other antibodies However optimal results are achieved if the protocol specific to the antibody is followed

biomapping

3 Take cell lysate sample (0 1ndash1 0 mL) and add 10 microL of antibody at appropriate dilution (refer to product specification)

4 Incubate for 90 minutes to overnight at 4 degC gently mixing the sample on a rotator

5 Add 50ndash100 microL of agarose conjugate suspension (25ndash50 microL agarosebed volume) Incubate for 15ndash60 minutes at 4 degC gently mixing the sample with a rotator

6 Centrifuge at 3000 3 g for 2 minutes at 4 degC and discard supernatant

7 Wash samples at least 4 times with 1 mL of washing buffer centrifuge at 3000 3 g for 2 minutes at 4 degC and discard the supernatant

Order 800-325-3010 Technical Service 800-325-5832 37

Preparation for SDS-PAGE

1 Resuspend each pellet in 25ndash100 microL of Laemmli sample buffer to a final concentration of 13 sample buffer Heat samples at 95 degC for 5 minutes

2 Centrifuge for 30 seconds at 12000 3 g at room temperature

3 Collect supernatant Samples can be loaded straight away or stored in sample buffer at ndash70 degC

Immunoblotting (Western Blotting)Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting It catalyzes the oxidation of luminol or TMB (33prime55prime-Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) Chemiluminescent detection is many times more sensitive than colorimetric but is more expensive and laborious Colorimetric detection can be performed on the bench and allows signal development to be observed directly

Immunodetection can be performed directly or indirectly Direct detection employs HRP-conjugated antibodies to the protein or tag whereas indirect is performed in two stages using an unlabeled lsquoprimaryrsquo antibody against the protein or tag and a labeled lsquosecondaryrsquo antibody against the first Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat No A7058 and Cat No H1029) and will give results for a wide spectrum of other antibodies However optimal results are achieved if the protocol specific to the antibody is followed

biomapping

Preparation for SDS-PAGE

1 Resuspend each pellet in 25ndash100 microL of Laemmli sample buffer to a final concentration of 13 sample buffer Heat samples at 95 degC for 5 minutes

2 Centrifuge for 30 seconds at 12000 3 g at room temperature

3 Collect supernatant Samples can be loaded straight away or stored in sample buffer at ndash70 degC

Immunoblotting (Western Blotting)Horseradish Peroxidase (HRP) is the most common enzyme system used for detection in immunoblotting It catalyzes the oxidation of luminol or TMB (33prime55prime-Tetramethylbenzidine) to yield a detectable signal in the form of light emission (chemiluminescent) or color development (colorimetric) Chemiluminescent detection is many times more sensitive than colorimetric but is more expensive and laborious Colorimetric detection can be performed on the bench and allows signal development to be observed directly

Immunodetection can be performed directly or indirectly Direct detection employs HRP-conjugated antibodies to the protein or tag whereas indirect is performed in two stages using an unlabeled lsquoprimaryrsquo antibody against the protein or tag and a labeled lsquosecondaryrsquo antibody against the first Indirect detection has a longer protocol but can offer greater sensitivity and be used to detect a greater range of proteins The following protocols are taken from labeled and unlabeled versions of monoclonal anti-polyhistidine antibody (Cat No A7058 and Cat No H1029) and will give results for a wide spectrum of other antibodies However optimal results are achieved if the protocol specific to the antibody is followed

biomapping

Order 800-325-3010 Technical Service 800-325-5832 39

Reagents

Transfer Buffer 1Tris-HCl pH 10 4 0 3 M

Methanol 20

Transfer buffer 2Tris-HCl pH 10 4 0 025 M

Methanol 20

Transfer buffer 3Tris-HCl pH 10 4 0 02 M

Methanol 20

e-amino caproic acid 25 mgmL

Blotting and detection buffersPBST PBS with 0 05 TWEEnreg 20 (Cat No P3563)

Ponceau S solution Cat No P7170Chemiluminescent or colorimetric detection reagents Cat Nos CPS160 or T0565

Protein transfer1 Build the transfer ldquosandwichrdquo onto the anode(+) plate as follows

bullTwo sheets blotting paper soaked in Transfer Buffer 1

bullOne sheet blotting paper soaked in Transfer Buffer 2

bullOne sheet of PVDF or nitrocellulose membrane pre-wet with deionized water Note If using PVDF pre-soak in 100 methanol for 10 seconds

bullProtein gel

bullThree sheets blotting paper soaked with Transfer Buffer 3

2 Transfer at 1 5 mAcm2 for 1 5 hours at room temperature

3 Remove the membrane from the apparatus and proceed with immunodetection

Optional Transfer can be verified by staining in Ponceau S solution for 2 minutes then rinsing with water prior to blocking

+ve

-ve

Direct Detection

1 Block the membrane using a solution of 5 non-fat dry milk in PBS for at least 60 minutes

2 Wash the membrane three times for 5 minutes in PBST

3 Incubate the membrane with anti-polyhistidine peroxidase conjugate (Cat No A7058) in PBST and 1 BSA for 2 hours Note Antibody dilution is usually 11000 but optimal concentration depends on the sample

4 Wash the membrane three times for 5 minutes in PBST

5 Treat the membrane with a peroxidase substrate and carry out detection

biomapping

Indirect Detection

1 Block the membrane using a solution of 5 non-fat dry milk in PBS for at least 60 minutes

2 Wash the membrane three times for 5 minutes in PBST

3 Incubate the membrane with anti-polyhistidine antibody (Cat No H1029) as the primary antibody in PBS containing 1 BSA for 2 hours Note Antibody dilution is usually 11000 but optimal concentration depends on the sample

4 Wash the membrane three times for 5 minutes in PBST

5 Incubate the membrane with anti-mouse IgG peroxidase conjugate (Cat No A9917) diluted 180000 in PBST for 60 minutes

6 Wash the membrane three times for 5 minutes in PBST

7 Treat the membrane with a peroxidase substrate and carry out detection

Order 800-325-3010 Technical Service 800-325-5832 41

biomapping

Immunohistochemistry Immunohistochemical staining is a valuable tool for detecting specific antigens in tissues Antibody reaction is detected enzymatically through use of HRP or alkaline phosphatase-conjugated secondary antibodies The procedure consists of a number of steps

bullParaffin removalrehydration

bullAntigen retrieval (optional if signal is weak)

bullInactivation of peroxidase (if HRP detection used)

bullPrimary Antibody Reaction

bullSecondary Antibody (or Extravidin) Reaction

bullDevelopment

bullCounterstaining

Reagents and Equipment

Formalin-fixed paraffin-embedded tissue sections10 mM phosphate buffered saline pH 7 4 (PBS) Cat No P3813 or P4417-tablet

Bovine serum albumin (BSA) Cat No A9647Diluent 1 BSA in PBS Cat No P3688Xylenes Cat No 95692Ethanol absolute Cat No E70230 1 Trypsin in PBS Trypsin tablet in 4 mM CaCl2 200 mm Tris pH 7 7 or 0 1 Protease in PBS

Cat No T7409 Cat No T7168-tablet Cat No P5380

Microwave antigen retrieval solution 10 mM sodium citrate Cat No C8532 pH 6 0 with 1 mM EDTA Cat No ED4SS pH 8 0

Secondary antibodyEnzyme substrate For peroxidase AEC Staining Kit Cat No AEC101

DAB Cat No D4418 For alkaline phosphatase Fast Red TRnapthol AS-MX Cat No F4648

Mayerrsquos hematoxylin Cat No MHS1Coplin staining jars Cat No S5641

Paraffin RemovalRehydration

1 Place the slides in a 56ndash60 degC oven for 15 minutes Caution Oven temperature must not exceed 60 degC

2 Transfer to a xylene bath and perform two changes of xylene for 5 minutes

3 Shake off excess liquid and rehydrate slides in two changes of fresh absolute ethanol for 3 minutes

4 Shake off excess liquid and place slides in fresh 90 ethanol for 3 minutes

5 Shake off excess liquid and place slides in fresh 80 ethanol for 3 minutes

6 Rinse the slides in gently running tap water for 30 seconds Note Avoid a direct jet which may wash off or loosen the section

7 Place in PBS wash bath for further rehydration (30 minutes at room temperature)

Order 800-325-3010 Technical Service 800-325-5832 43

Antigen Retrieval (optional step for weak signal)Protein epitopes are less accessible in fixed tissues than in solution so occasionally an antigen ldquounmaskingrdquo step by enzyme digestion may be required

Note This step is performed only in cases where weak or no staining occurs or for antigens requiring ldquounmaskingrdquo according to the primary antibody specifications There are several possible ways for retrieval depending on the antibody and the antigen

Enzymatic method

1 Apply 0 1 trypsin in PBS or 0 1 protease in PBS for 2ndash30 minutes at 37 degC Note Extending the incubation time may also enhance specific staining

2 Rinse in PBS for 10 minutes

Microwave retrieval

1 Wash the slides with deionized H2O and place them in a microwave-resistant plastic staining jar containing antigen retrieval solution Note Make sure slides are fully covered with solution

2 Operate the microwave oven for 5 minutes on high power (~700 watts) Note Make sure slides are still covered with retrieval solution or add fresh solution and repeat microwaving

3 This process can be repeated 2ndash3 times

4 Let cool slowly at room temperature for at least 20 minutes before proceeding to the next step

biomapping

Inactivation of Peroxidase (if HRP detection used)Note This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase

1 Place the slides on a flat level surface Note Do not allow slides to touch each other or dry out at any time

2 Add drops of 3 hydrogen peroxide to cover the whole section

3 Incubate for 5 minutes at room temperature

4 Rinse with PBS from a wash bottle

5 Place the slide in PBS wash bath for 2 minutes

Order 800-325-3010 Technical Service 800-325-5832 45

Primary Antibody ReactionPre-incubation of the sample with 5 BSA for 10 minutes prior to the primary antibody reaction may decrease background staining For best results with animal tissues use 5ndash10 normal serum from the same species as the host of the secondary antibody

Optimal dilution and incubation times should be determined for each primary antibody prior to use

1 Allow the slides to drain shake off excess fluid and carefully wipe each slide avoiding the sections

2 Dilute the primary antibody or negative control reagent in diluent Note The diluent alone may be used as a negative control A positive control slide (a tissue known to contain the antigen under study) should also be run

3 Apply 100 microL of primary antibody solution to the appropriate slides covering the tissue sections

4 Tilt each slide in two different directions so the liquid is spread evenly

5 Incubate for at least 60 minutes at 37 degC in humidified chamber Note Longer incubations are advised for low density antigens

6 Rinse gently with PBS from a wash bottle Place the slide in a PBS wash bath for 5 minutes

biomapping

Secondary ReactionOption 1 BiotinStreptavidin Detection

1 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the biotinylated secondary antibody in diluent to its optimal concentration

3 Apply 100 microL to each slide covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate in a humidity chamber for at least 30 minutes at room temperature

6 Rinse gently with PBS from a wash bottle

7 Place the slide in a PBS wash bath for 5 minutes

8 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

Order 800-325-3010 Technical Service 800-325-5832 47

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Antigen Retrieval (optional step for weak signal)Protein epitopes are less accessible in fixed tissues than in solution so occasionally an antigen ldquounmaskingrdquo step by enzyme digestion may be required

Note This step is performed only in cases where weak or no staining occurs or for antigens requiring ldquounmaskingrdquo according to the primary antibody specifications There are several possible ways for retrieval depending on the antibody and the antigen

Enzymatic method

1 Apply 0 1 trypsin in PBS or 0 1 protease in PBS for 2ndash30 minutes at 37 degC Note Extending the incubation time may also enhance specific staining

2 Rinse in PBS for 10 minutes

Microwave retrieval

1 Wash the slides with deionized H2O and place them in a microwave-resistant plastic staining jar containing antigen retrieval solution Note Make sure slides are fully covered with solution

2 Operate the microwave oven for 5 minutes on high power (~700 watts) Note Make sure slides are still covered with retrieval solution or add fresh solution and repeat microwaving

3 This process can be repeated 2ndash3 times

4 Let cool slowly at room temperature for at least 20 minutes before proceeding to the next step

biomapping

Inactivation of Peroxidase (if HRP detection used)Note This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase

1 Place the slides on a flat level surface Note Do not allow slides to touch each other or dry out at any time

2 Add drops of 3 hydrogen peroxide to cover the whole section

3 Incubate for 5 minutes at room temperature

4 Rinse with PBS from a wash bottle

5 Place the slide in PBS wash bath for 2 minutes

Order 800-325-3010 Technical Service 800-325-5832 45

Primary Antibody ReactionPre-incubation of the sample with 5 BSA for 10 minutes prior to the primary antibody reaction may decrease background staining For best results with animal tissues use 5ndash10 normal serum from the same species as the host of the secondary antibody

Optimal dilution and incubation times should be determined for each primary antibody prior to use

1 Allow the slides to drain shake off excess fluid and carefully wipe each slide avoiding the sections

2 Dilute the primary antibody or negative control reagent in diluent Note The diluent alone may be used as a negative control A positive control slide (a tissue known to contain the antigen under study) should also be run

3 Apply 100 microL of primary antibody solution to the appropriate slides covering the tissue sections

4 Tilt each slide in two different directions so the liquid is spread evenly

5 Incubate for at least 60 minutes at 37 degC in humidified chamber Note Longer incubations are advised for low density antigens

6 Rinse gently with PBS from a wash bottle Place the slide in a PBS wash bath for 5 minutes

biomapping

Secondary ReactionOption 1 BiotinStreptavidin Detection

1 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the biotinylated secondary antibody in diluent to its optimal concentration

3 Apply 100 microL to each slide covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate in a humidity chamber for at least 30 minutes at room temperature

6 Rinse gently with PBS from a wash bottle

7 Place the slide in a PBS wash bath for 5 minutes

8 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

Order 800-325-3010 Technical Service 800-325-5832 47

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Inactivation of Peroxidase (if HRP detection used)Note This step is performed only when using peroxidase-conjugated secondary antibodies or ExtrAvidin-Peroxidase

1 Place the slides on a flat level surface Note Do not allow slides to touch each other or dry out at any time

2 Add drops of 3 hydrogen peroxide to cover the whole section

3 Incubate for 5 minutes at room temperature

4 Rinse with PBS from a wash bottle

5 Place the slide in PBS wash bath for 2 minutes

Order 800-325-3010 Technical Service 800-325-5832 45

Primary Antibody ReactionPre-incubation of the sample with 5 BSA for 10 minutes prior to the primary antibody reaction may decrease background staining For best results with animal tissues use 5ndash10 normal serum from the same species as the host of the secondary antibody

Optimal dilution and incubation times should be determined for each primary antibody prior to use

1 Allow the slides to drain shake off excess fluid and carefully wipe each slide avoiding the sections

2 Dilute the primary antibody or negative control reagent in diluent Note The diluent alone may be used as a negative control A positive control slide (a tissue known to contain the antigen under study) should also be run

3 Apply 100 microL of primary antibody solution to the appropriate slides covering the tissue sections

4 Tilt each slide in two different directions so the liquid is spread evenly

5 Incubate for at least 60 minutes at 37 degC in humidified chamber Note Longer incubations are advised for low density antigens

6 Rinse gently with PBS from a wash bottle Place the slide in a PBS wash bath for 5 minutes

biomapping

Secondary ReactionOption 1 BiotinStreptavidin Detection

1 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the biotinylated secondary antibody in diluent to its optimal concentration

3 Apply 100 microL to each slide covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate in a humidity chamber for at least 30 minutes at room temperature

6 Rinse gently with PBS from a wash bottle

7 Place the slide in a PBS wash bath for 5 minutes

8 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

Order 800-325-3010 Technical Service 800-325-5832 47

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Primary Antibody ReactionPre-incubation of the sample with 5 BSA for 10 minutes prior to the primary antibody reaction may decrease background staining For best results with animal tissues use 5ndash10 normal serum from the same species as the host of the secondary antibody

Optimal dilution and incubation times should be determined for each primary antibody prior to use

1 Allow the slides to drain shake off excess fluid and carefully wipe each slide avoiding the sections

2 Dilute the primary antibody or negative control reagent in diluent Note The diluent alone may be used as a negative control A positive control slide (a tissue known to contain the antigen under study) should also be run

3 Apply 100 microL of primary antibody solution to the appropriate slides covering the tissue sections

4 Tilt each slide in two different directions so the liquid is spread evenly

5 Incubate for at least 60 minutes at 37 degC in humidified chamber Note Longer incubations are advised for low density antigens

6 Rinse gently with PBS from a wash bottle Place the slide in a PBS wash bath for 5 minutes

biomapping

Secondary ReactionOption 1 BiotinStreptavidin Detection

1 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the biotinylated secondary antibody in diluent to its optimal concentration

3 Apply 100 microL to each slide covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate in a humidity chamber for at least 30 minutes at room temperature

6 Rinse gently with PBS from a wash bottle

7 Place the slide in a PBS wash bath for 5 minutes

8 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

Order 800-325-3010 Technical Service 800-325-5832 47

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Secondary ReactionOption 1 BiotinStreptavidin Detection

1 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the biotinylated secondary antibody in diluent to its optimal concentration

3 Apply 100 microL to each slide covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate in a humidity chamber for at least 30 minutes at room temperature

6 Rinse gently with PBS from a wash bottle

7 Place the slide in a PBS wash bath for 5 minutes

8 Allow the slides to drain shake off excess fluid and carefully wipe the slide as before

Order 800-325-3010 Technical Service 800-325-5832 47

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

9 Dilute StreptavidinExtrAvidinreg conjugate in diluent to its optimal concentration

10 Apply 100 microL to all slides covering the sections

11 Tilt each slide in two different directions

12 Incubate in humidified chamber for at least 20 minutes at room temperature

13 Rinse gently with PBS from a wash bottle

14 Place the slide in PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during this final wash step

15 Skip to Development step

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Option 2 Enzyme-labeled Secondary Antibody

1 Allow the slide to drain shake off excess fluid and carefully wipe the slide as before

2 Dilute the peroxidase or phosphatase conjugated secondary antibody in the diluent

3 Apply 100 microL to all slides covering the tissue sections

4 Tilt each slide in two different directions

5 Incubate 30 minutes at room temperature or at 37 degC in humidified chamber

6 Rinse gently with PBS from a wash bottle

7 Place the slides in a PBS wash bath for 5 minutes Note It is recommended to prepare the detection substrate mixture during the final wash step

Order 800-325-3010 Technical Service 800-325-5832 49

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

DevelopmentNote Addition of 1 mM levamisole (Cat No L9756) to alkaline phosphatase substrate solution will largely inhibit endogenous alkaline phosphatase activity (except that of the intestinal isoenzyme)

1 Allow each slide to drain shake off excess fluid and carefully wipe the slide as before

2 Apply enough drops of freshly prepared substrate mixture to cover the tissue section

3 Incubate for 5ndash10 minutes or until desired color reaction is observed when monitored with the microscope Terminate the reaction before background staining appears in the negative controls by rinsing gently with distilled water from a wash bottle

Counterstaining

Note When using AEC substrate do not use alcohol-containing solutions for counterstaining (e g Harrisrsquo hematoxylin acid alcohol) since the AEC stain formed by this method is soluble in organic solvents The slide must not be dehydrated brought back to toluene (or xylene) or mounted in toluene-containing mountants

1 Apply enough Mayerrsquos hematoxylin to cover the section or place the slide in a bath of Mayerrsquos hematoxylin

2 Incubate for 30 seconds to 5 minutes depending on strength of the hematoxylin

biomapping

Order 800-325-3010 Technical Service 800-325-5832 51

3 Rinse the slide gently with distilled water from a wash bottle

4 Rinse the slide under gently running tap water for 5 minutes Note Avoid a direct jet which may wash off or loosen the section

5 Mount the sections using an aqueous mounting medium such as glycerol gelatin Coverslip may be sealed with clear nail polish

Immunofluorescence Immunostaining is a very versatile technique and if the antigen is highly localized it can detect as few as a thousand antigen molecules in a cell In some circumstances it can also be used to determine the approximate concentration of an antigen especially by an image analyzer

As a first step cells to be stained are attached to a solid support to allow easy handling in subsequent procedures This can be achieved by several methods adherent cells may be grown on microscope slides coverslips or an optically suitable plastic support Suspension cells can be centrifuged onto glass slides bound to solid support using chemical linkers or in some cases handled in suspension

The second step is to fix and permeabilize the cells to ensure free access of the antibody to its antigen Perfect fixation would immobilize the antigens while retaining authentic cellular and subcellular architecture permitting unhindered access of antibodies to all cells and subcellular compartments The correct choice of method will depend on the nature of the antigen being examined and on the properties of the antibody used but fixation methods fall generally into two classes organic solvents and crosslinking reagents Organic solvents such as alcohols and acetone remove lipids and dehydrate the cells while precipitating the proteins on the cellular architecture Crosslinking reagents (such as paraformaldehyde) form intermolecular bridges normally through free amino groups thus creating a network of linked antigens Crosslinkers preserve cell structure better than organic solvents but may reduce the antigenicity of some cell components and require the addition of a permeabilization step to allow access of the antibody to the specimen

Either method may denature protein antigens and for this reason antibodies prepared against denatured proteins may be more useful for cell staining Four different fixation methods are described the appropriate fixation method should be chosen according to the relevant application

biomapping

The third step of cell staining involves incubation of cell preparations with the antibody Unbound antibody is removed by washing and the bound antibody is detected either directly (if the primary antibody is labeled) or indirectly using a fluorophore-labeled secondary reagent Finally the staining is evaluated using fluorescence microscopy

Reagents and Equipment

PBS 0 01 M Phosphate buffered saline pH 7 2ndash7 4 Cat No P3813 or P4417

Methanol cooled at ndash20 degC for at least 1 hour Acetone cooled at ndash20 degC for at least1 hour

Cat No 32213 Cat No 24201

3ndash4 Paraformaldehyde in PBS with 0 5 TRITOnreg X-100 in PBS

Cat No P6148 Dissolved in PBS using 5 M naOH and mild heating Cat No T9284

3ndash4 Paraformaldehyde in PBS Methanol cooled at ndash20 degC for at least 1 hour

Cat No P6148 Dissolved in PBS using 5 M naOH and mild heating Cat No 32213

PEM Buffer Ethanol cooled at ndash20 degC for at least 1 hour 0 1 M PIPES with 5 mM EGTA and 2 mM MgCl2 middot 6 H2O Bring to pH 6 8 using naOH solution Cat No 270741

Primary antibodyAntibody controlsSecondary antibody-fluorophore-labeledAqueous mounting mediumMicroscope glass slides 76 x 25 mm Cat No S8902Fluorescence microscope equipped with appropriate filter for fluorescence detectionInverted light microscope

Notes This is provided as a general protocol Optimal dilutions for the primary and secondary anti-bodies cell preparation controls as well as incubation times will need to be determined empirically and may require extensive titration Ideally one would use the primary antibody as recommended in the product data sheet The appropriate negative and positive controls should also be included

Protect fluorescent conjugates and labeled slides from the light Incubate samples in the dark and cover whenever possible

biomapping

The third step of cell staining involves incubation of cell preparations with the antibody Unbound antibody is removed by washing and the bound antibody is detected either directly (if the primary antibody is labeled) or indirectly using a fluorophore-labeled secondary reagent Finally the staining is evaluated using fluorescence microscopy

Reagents and Equipment

PBS 0 01 M Phosphate buffered saline pH 7 2ndash7 4 Cat No P3813 or P4417

Methanol cooled at ndash20 degC for at least 1 hour Acetone cooled at ndash20 degC for at least1 hour

Cat No 32213 Cat No 24201

3ndash4 Paraformaldehyde in PBS with 0 5 TRITOnreg X-100 in PBS

Cat No P6148 Dissolved in PBS using 5 M naOH and mild heating Cat No T9284

3ndash4 Paraformaldehyde in PBS Methanol cooled at ndash20 degC for at least 1 hour

Cat No P6148 Dissolved in PBS using 5 M naOH and mild heating Cat No 32213

PEM Buffer Ethanol cooled at ndash20 degC for at least 1 hour 0 1 M PIPES with 5 mM EGTA and 2 mM MgCl2 middot 6 H2O Bring to pH 6 8 using naOH solution Cat No 270741

Primary antibodyAntibody controlsSecondary antibody-fluorophore-labeledAqueous mounting mediumMicroscope glass slides 76 x 25 mm Cat No S8902Fluorescence microscope equipped with appropriate filter for fluorescence detectionInverted light microscope

Notes This is provided as a general protocol Optimal dilutions for the primary and secondary anti-bodies cell preparation controls as well as incubation times will need to be determined empirically and may require extensive titration Ideally one would use the primary antibody as recommended in the product data sheet The appropriate negative and positive controls should also be included

Protect fluorescent conjugates and labeled slides from the light Incubate samples in the dark and cover whenever possible

Order 800-325-3010 Technical Service 800-325-5832 53

Cell Preparation 1 Remove cells from incubator Inspect under inverted light microscope to verify the desired

appearance Discard medium

2 Rinse with PBS remove excess solution

Fixation Four different fixation methods are described Choose the appropriate fixation method according to the application (or product data sheet recommendation)

Methanol-Acetone Fixation

1 Fix in cooled methanol 10 minutes at ndash20 degC

2 Remove excess methanol

3 Permeabilize with cooled acetone for 1 minutes at ndash20 degC

Paraformaldehyde-Tritonreg Fixation

1 Fix in 3ndash4 paraformaldehyde for 10ndash20 minutes

2 Rinse briefly with PBS

3 Permeabilize with 0 5 TRITOn X-100 for 2ndash10 minutes

Paraformaldehyde-Methanol Fixation

1 Fix in 3ndash4 paraformaldehyde for 10ndash20 minutes

2 Rinse briefly with PBS

3 Permeabilize with cooled methanol for 5ndash10 minutes at ndash20 degC

PEM-Ethanol Fixation

1 Fix in PEM buffer for 10 minutes

2 Rinse twice briefly with PBS

3 Permeabilize with cooled ethanol for 5ndash10 minutes at ndash20 degC

4 Wash three times (at least 5 minutes each) with PBS

biomapping

Application of Primary Antibody

1 Dilute primary antibody in PBS to appropriate dilution Apply on coverslips over the cells and incubate for 60 minutes at room temperature Note A humidified chamber is recommended

2 Wash three times for at least 5 minutes with PBS

Application of Secondary Antibody Note Secondary antibody is applied only in indirect assays

1 Dilute labeled secondary antibody to appropriate dilution in PBS Apply on coverslips and incubate for 30 minutes at room temperature

2 Wash three times (at least 5 minutes each) with PBS

3 Remove excess PBS

EvaluationIt is advisable to run the appropriate negative controls negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies The ideal negative control reagent is a fluorophore-conjugated mouse monoclonal or myeloma protein It should be isotype-matched not specific for cells of the species being studied and of the same concentration as the test antibody The degree of autofluorescence or negative control reagent fluorescence will vary with the type of cells under study and the sensitivity of the instrument used For fluorescent analysis of cells with Fc receptors the use of isotype-matched negative controls is mandatory

1 Mount coverslips with mounting medium and invert onto glass slides

2 Inspect under the microscope and photograph

Order 800-325-3010 Technical Service 800-325-5832 55

biomapping

Detection substrate Alkaline phosphatase SIGMAFASTtrade pnPP tablets (Cat No N1891) HRP SIGMAFASTtrade OPD tablets (Cat No P9187)

Stopping reagent (optional) Alkaline phosphatase 3 M naOH HRP 3 M HCl or 3 M H2SO4

Multiwell plates (Cat No CLS9017)

ELISAEnzyme-Linked Immunosorbent Assay (ELISA) is a method for specific quantitation of compounds Its specificity flexibility and reproducibility (as well as low cost) have lead to its widespread use as a diagnostic tool In a protein research setting it acts as a convenient method for screening levels of cellular proteins e g expression profiling

Indirect ELISA

Reagents and Equipment

Phosphate buffered saline (PBS) tablet 10 mM phosphate buffer pH 7 4 150 mM naCl and 0 1 sodium azide

Carbonate-Bicarbonate buffer capsule pH 9 6 (Cat No C3041)

Washing buffer (PBST) 10 mM phosphate buffer pH 7 4 150 mM naCl 0 05 TWEEnreg 20 (Cat No P3563)

Antibody controls Species and isotype-matched non-specific immunoglobulin (e g mouse myeloma proteins as a control for a mouse monoclonal primary antibody)

Secondary antibody Either alkaline phosphatase or horseradish peroxidase (HRP)-conjugate

Paraformaldehyde-Methanol Fixation

1 Fix in 3ndash4 paraformaldehyde for 10ndash20 minutes

2 Rinse briefly with PBS

3 Permeabilize with cooled methanol for 5ndash10 minutes at ndash20 degC

PEM-Ethanol Fixation

1 Fix in PEM buffer for 10 minutes

2 Rinse twice briefly with PBS

3 Permeabilize with cooled ethanol for 5ndash10 minutes at ndash20 degC

4 Wash three times (at least 5 minutes each) with PBS

biomapping

Application of Primary Antibody

1 Dilute primary antibody in PBS to appropriate dilution Apply on coverslips over the cells and incubate for 60 minutes at room temperature Note A humidified chamber is recommended

2 Wash three times for at least 5 minutes with PBS

Application of Secondary Antibody Note Secondary antibody is applied only in indirect assays

1 Dilute labeled secondary antibody to appropriate dilution in PBS Apply on coverslips and incubate for 30 minutes at room temperature

2 Wash three times (at least 5 minutes each) with PBS

3 Remove excess PBS

EvaluationIt is advisable to run the appropriate negative controls negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies The ideal negative control reagent is a fluorophore-conjugated mouse monoclonal or myeloma protein It should be isotype-matched not specific for cells of the species being studied and of the same concentration as the test antibody The degree of autofluorescence or negative control reagent fluorescence will vary with the type of cells under study and the sensitivity of the instrument used For fluorescent analysis of cells with Fc receptors the use of isotype-matched negative controls is mandatory

1 Mount coverslips with mounting medium and invert onto glass slides

2 Inspect under the microscope and photograph

Order 800-325-3010 Technical Service 800-325-5832 55

biomapping

Detection substrate Alkaline phosphatase SIGMAFASTtrade pnPP tablets (Cat No N1891) HRP SIGMAFASTtrade OPD tablets (Cat No P9187)

Stopping reagent (optional) Alkaline phosphatase 3 M naOH HRP 3 M HCl or 3 M H2SO4

Multiwell plates (Cat No CLS9017)

ELISAEnzyme-Linked Immunosorbent Assay (ELISA) is a method for specific quantitation of compounds Its specificity flexibility and reproducibility (as well as low cost) have lead to its widespread use as a diagnostic tool In a protein research setting it acts as a convenient method for screening levels of cellular proteins e g expression profiling

Indirect ELISA

Reagents and Equipment

Phosphate buffered saline (PBS) tablet 10 mM phosphate buffer pH 7 4 150 mM naCl and 0 1 sodium azide

Carbonate-Bicarbonate buffer capsule pH 9 6 (Cat No C3041)

Washing buffer (PBST) 10 mM phosphate buffer pH 7 4 150 mM naCl 0 05 TWEEnreg 20 (Cat No P3563)

Antibody controls Species and isotype-matched non-specific immunoglobulin (e g mouse myeloma proteins as a control for a mouse monoclonal primary antibody)

Secondary antibody Either alkaline phosphatase or horseradish peroxidase (HRP)-conjugate

Application of Primary Antibody

1 Dilute primary antibody in PBS to appropriate dilution Apply on coverslips over the cells and incubate for 60 minutes at room temperature Note A humidified chamber is recommended

2 Wash three times for at least 5 minutes with PBS

Application of Secondary Antibody Note Secondary antibody is applied only in indirect assays

1 Dilute labeled secondary antibody to appropriate dilution in PBS Apply on coverslips and incubate for 30 minutes at room temperature

2 Wash three times (at least 5 minutes each) with PBS

3 Remove excess PBS

EvaluationIt is advisable to run the appropriate negative controls negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies The ideal negative control reagent is a fluorophore-conjugated mouse monoclonal or myeloma protein It should be isotype-matched not specific for cells of the species being studied and of the same concentration as the test antibody The degree of autofluorescence or negative control reagent fluorescence will vary with the type of cells under study and the sensitivity of the instrument used For fluorescent analysis of cells with Fc receptors the use of isotype-matched negative controls is mandatory

1 Mount coverslips with mounting medium and invert onto glass slides

2 Inspect under the microscope and photograph

Order 800-325-3010 Technical Service 800-325-5832 55

biomapping

Detection substrate Alkaline phosphatase SIGMAFASTtrade pnPP tablets (Cat No N1891) HRP SIGMAFASTtrade OPD tablets (Cat No P9187)

Stopping reagent (optional) Alkaline phosphatase 3 M naOH HRP 3 M HCl or 3 M H2SO4

Multiwell plates (Cat No CLS9017)

ELISAEnzyme-Linked Immunosorbent Assay (ELISA) is a method for specific quantitation of compounds Its specificity flexibility and reproducibility (as well as low cost) have lead to its widespread use as a diagnostic tool In a protein research setting it acts as a convenient method for screening levels of cellular proteins e g expression profiling

Indirect ELISA

Reagents and Equipment

Phosphate buffered saline (PBS) tablet 10 mM phosphate buffer pH 7 4 150 mM naCl and 0 1 sodium azide

Carbonate-Bicarbonate buffer capsule pH 9 6 (Cat No C3041)

Washing buffer (PBST) 10 mM phosphate buffer pH 7 4 150 mM naCl 0 05 TWEEnreg 20 (Cat No P3563)

Antibody controls Species and isotype-matched non-specific immunoglobulin (e g mouse myeloma proteins as a control for a mouse monoclonal primary antibody)

Secondary antibody Either alkaline phosphatase or horseradish peroxidase (HRP)-conjugate

biomapping

Detection substrate Alkaline phosphatase SIGMAFASTtrade pnPP tablets (Cat No N1891) HRP SIGMAFASTtrade OPD tablets (Cat No P9187)

Stopping reagent (optional) Alkaline phosphatase 3 M naOH HRP 3 M HCl or 3 M H2SO4

Multiwell plates (Cat No CLS9017)

ELISAEnzyme-Linked Immunosorbent Assay (ELISA) is a method for specific quantitation of compounds Its specificity flexibility and reproducibility (as well as low cost) have lead to its widespread use as a diagnostic tool In a protein research setting it acts as a convenient method for screening levels of cellular proteins e g expression profiling

Indirect ELISA

Reagents and Equipment

Phosphate buffered saline (PBS) tablet 10 mM phosphate buffer pH 7 4 150 mM naCl and 0 1 sodium azide

Carbonate-Bicarbonate buffer capsule pH 9 6 (Cat No C3041)

Washing buffer (PBST) 10 mM phosphate buffer pH 7 4 150 mM naCl 0 05 TWEEnreg 20 (Cat No P3563)

Antibody controls Species and isotype-matched non-specific immunoglobulin (e g mouse myeloma proteins as a control for a mouse monoclonal primary antibody)

Secondary antibody Either alkaline phosphatase or horseradish peroxidase (HRP)-conjugate

Antigen Coating

1 Prepare an antigen solution at the appropriate concentration in carbonate-bicarbonate buffer or PBS

2 Pipette 0 2 mL of the above solution into each well of the multiwell plate

3 Incubate at 37 degC for 30 minutes or incubate (covered) overnight at 4 degC

4 Remove the coating solution Wash three times with PBST

Note If problems with non-specific binding occur an additional blocking step (30 minutes with 5 BSA-PBS) may be required For further information see Vogt R F et al (1987)J Immunol Meth 101 43

Order 800-325-3010 Technical Service 800-325-5832 57

Primary Antibody Reaction

1 Dilute the monoclonal primary antibody in PBST The optimal dilution should be determined using a titration assay

2 Add 0 2 mL of the diluted monoclonal antibody to each well The negative control should be species and isotype-matched non-specific immunoglobulin diluted in PBST

3 Incubate at room temperature for 2 hours

4 Wash as in step 4 of Antigen Coating

Application of Secondary Antibody

1 Dilute the enzyme-conjugated secondary antibody in PBST Add 0 2 mL of this solution to each well The optimal dilution should be determined using a titration assay

2 Incubate at room temperature for 2 hours

3 Wash as in step 4 of Antigen Coating

Note During the last incubation and immediately before use prepare the enzyme substrate or bring the pre-made liquid substrate to room temperature

biomapping

59Order 800-325-3010 Technical Service 800-325-5832

Development

1 Add 0 2 mL of the freshly prepared substrate to each well

2 Color should develop in positive wells after 30 minutes (yellow and orange for pnPP and OPD respectively) Note Absorbance may be read directly in a multiwell platereader (at 405 nm and 450 nm for pNPP and OPD respectively) or the reaction may be stopped with 50 microl per well of the appropriate stopping reagent and absorbance read later

References 1 Beesley J E (ed ) (1993) ldquoImmunocytochemistry A Practical Approachrdquo IRL Press 215 21(Prod no Z35005-2)

2 Bullock G R and Petrusz P (eds ) (1982) (1983) (1985) (1989) ldquoTechniques in Immunocytochemistryrdquo Volumes 1 2 3 and 4 Academic Press See Vol 1 18

3 Coligan J E et al (eds ) (1991) ldquoCurrent Protocols in Immunology rdquo John Wiley amp Sons (new York) 2 1 1

4 Crowther J R (1995) ldquoMethods in Molecular Biology Volume 42 ELISA Theory and Practicerdquo Humana Press (new Jersey) (Prod no Z36415-0)

5 Cuello A C (ed ) (1993) ldquoImmunohistochemistry IIrdquo Wiley Press (new York) (Prod no I4018)

6 Desphande S S (1996) ldquoEnzyme Immunoassays From Concept to Product Developmentrdquo Chapman amp Hall (new York) (Prod no Z37025-8)

7 Diamandis E P and Christopoulos T K (eds ) (1996) ldquoImmunoassayrdquo Academic Press (new York) (Prod no Z37367-2)

8 Ferencik M (1993) ldquoHandbook of Immunochemistryrdquo Chapman amp Hall (new York) 346ndash356 (Prod no Z37020-7)

9 Giloh H and Sedat J W (1982) ldquoFluorescence Microscopy Reduced Photobleaching of Rhodamine and Fluorescein Protein Conjugates by n-Propyl Gallate rdquo Science 217 1252ndash1255

10 Harlow E and Lane D (1988) ldquoAntibodies A Laboratory Manual rdquo Cold Spring Harbor Laboratory Press (new York) 579 (Cat No A2926)

11 Johnson G D et al (1982) ldquoFading of Immunofluorescence During Microscopy A Study of the Phenomenon and Its Remedy rdquo J Immunol Methods 55 231ndash242

12 Longin A et al (1993) ldquoComparison of Anti-Fading Agents Used in Fluorescence Microscopy Image Analysis and Laser Confocal Microscopy Study rdquo J Histochem Cytochem 41(12) 1833ndash1840

13 P Tijssen (1985) ldquoLaboratory Techniques in Biochemistry and Molecular Biology Practice and Theory of Enzyme Immunoassay rdquo Elsevier Science Publishers (Amsterdam The netherlands)

14 Storz H and Jelke E (1984) ldquoPhotomicrography of Weakly Fluorescent ObjectsmdashEmployment of p-phenylenediamine as a Blocker of Fading rdquo Acta Histochem 75 133ndash139

Primary Antibody Reaction

1 Dilute the monoclonal primary antibody in PBST The optimal dilution should be determined using a titration assay

2 Add 0 2 mL of the diluted monoclonal antibody to each well The negative control should be species and isotype-matched non-specific immunoglobulin diluted in PBST

3 Incubate at room temperature for 2 hours

4 Wash as in step 4 of Antigen Coating

Application of Secondary Antibody

1 Dilute the enzyme-conjugated secondary antibody in PBST Add 0 2 mL of this solution to each well The optimal dilution should be determined using a titration assay

2 Incubate at room temperature for 2 hours

3 Wash as in step 4 of Antigen Coating

Note During the last incubation and immediately before use prepare the enzyme substrate or bring the pre-made liquid substrate to room temperature

biomapping

59Order 800-325-3010 Technical Service 800-325-5832

Development

1 Add 0 2 mL of the freshly prepared substrate to each well

2 Color should develop in positive wells after 30 minutes (yellow and orange for pnPP and OPD respectively) Note Absorbance may be read directly in a multiwell platereader (at 405 nm and 450 nm for pNPP and OPD respectively) or the reaction may be stopped with 50 microl per well of the appropriate stopping reagent and absorbance read later

References 1 Beesley J E (ed ) (1993) ldquoImmunocytochemistry A Practical Approachrdquo IRL Press 215 21(Prod no Z35005-2)

2 Bullock G R and Petrusz P (eds ) (1982) (1983) (1985) (1989) ldquoTechniques in Immunocytochemistryrdquo Volumes 1 2 3 and 4 Academic Press See Vol 1 18

3 Coligan J E et al (eds ) (1991) ldquoCurrent Protocols in Immunology rdquo John Wiley amp Sons (new York) 2 1 1

4 Crowther J R (1995) ldquoMethods in Molecular Biology Volume 42 ELISA Theory and Practicerdquo Humana Press (new Jersey) (Prod no Z36415-0)

5 Cuello A C (ed ) (1993) ldquoImmunohistochemistry IIrdquo Wiley Press (new York) (Prod no I4018)

6 Desphande S S (1996) ldquoEnzyme Immunoassays From Concept to Product Developmentrdquo Chapman amp Hall (new York) (Prod no Z37025-8)

7 Diamandis E P and Christopoulos T K (eds ) (1996) ldquoImmunoassayrdquo Academic Press (new York) (Prod no Z37367-2)

8 Ferencik M (1993) ldquoHandbook of Immunochemistryrdquo Chapman amp Hall (new York) 346ndash356 (Prod no Z37020-7)

9 Giloh H and Sedat J W (1982) ldquoFluorescence Microscopy Reduced Photobleaching of Rhodamine and Fluorescein Protein Conjugates by n-Propyl Gallate rdquo Science 217 1252ndash1255

10 Harlow E and Lane D (1988) ldquoAntibodies A Laboratory Manual rdquo Cold Spring Harbor Laboratory Press (new York) 579 (Cat No A2926)

11 Johnson G D et al (1982) ldquoFading of Immunofluorescence During Microscopy A Study of the Phenomenon and Its Remedy rdquo J Immunol Methods 55 231ndash242

12 Longin A et al (1993) ldquoComparison of Anti-Fading Agents Used in Fluorescence Microscopy Image Analysis and Laser Confocal Microscopy Study rdquo J Histochem Cytochem 41(12) 1833ndash1840

13 P Tijssen (1985) ldquoLaboratory Techniques in Biochemistry and Molecular Biology Practice and Theory of Enzyme Immunoassay rdquo Elsevier Science Publishers (Amsterdam The netherlands)

14 Storz H and Jelke E (1984) ldquoPhotomicrography of Weakly Fluorescent ObjectsmdashEmployment of p-phenylenediamine as a Blocker of Fading rdquo Acta Histochem 75 133ndash139

59Order 800-325-3010 Technical Service 800-325-5832

Development

1 Add 0 2 mL of the freshly prepared substrate to each well

2 Color should develop in positive wells after 30 minutes (yellow and orange for pnPP and OPD respectively) Note Absorbance may be read directly in a multiwell platereader (at 405 nm and 450 nm for pNPP and OPD respectively) or the reaction may be stopped with 50 microl per well of the appropriate stopping reagent and absorbance read later

References 1 Beesley J E (ed ) (1993) ldquoImmunocytochemistry A Practical Approachrdquo IRL Press 215 21(Prod no Z35005-2)

2 Bullock G R and Petrusz P (eds ) (1982) (1983) (1985) (1989) ldquoTechniques in Immunocytochemistryrdquo Volumes 1 2 3 and 4 Academic Press See Vol 1 18

3 Coligan J E et al (eds ) (1991) ldquoCurrent Protocols in Immunology rdquo John Wiley amp Sons (new York) 2 1 1

4 Crowther J R (1995) ldquoMethods in Molecular Biology Volume 42 ELISA Theory and Practicerdquo Humana Press (new Jersey) (Prod no Z36415-0)

5 Cuello A C (ed ) (1993) ldquoImmunohistochemistry IIrdquo Wiley Press (new York) (Prod no I4018)

6 Desphande S S (1996) ldquoEnzyme Immunoassays From Concept to Product Developmentrdquo Chapman amp Hall (new York) (Prod no Z37025-8)

7 Diamandis E P and Christopoulos T K (eds ) (1996) ldquoImmunoassayrdquo Academic Press (new York) (Prod no Z37367-2)

8 Ferencik M (1993) ldquoHandbook of Immunochemistryrdquo Chapman amp Hall (new York) 346ndash356 (Prod no Z37020-7)

9 Giloh H and Sedat J W (1982) ldquoFluorescence Microscopy Reduced Photobleaching of Rhodamine and Fluorescein Protein Conjugates by n-Propyl Gallate rdquo Science 217 1252ndash1255

10 Harlow E and Lane D (1988) ldquoAntibodies A Laboratory Manual rdquo Cold Spring Harbor Laboratory Press (new York) 579 (Cat No A2926)

11 Johnson G D et al (1982) ldquoFading of Immunofluorescence During Microscopy A Study of the Phenomenon and Its Remedy rdquo J Immunol Methods 55 231ndash242

12 Longin A et al (1993) ldquoComparison of Anti-Fading Agents Used in Fluorescence Microscopy Image Analysis and Laser Confocal Microscopy Study rdquo J Histochem Cytochem 41(12) 1833ndash1840

13 P Tijssen (1985) ldquoLaboratory Techniques in Biochemistry and Molecular Biology Practice and Theory of Enzyme Immunoassay rdquo Elsevier Science Publishers (Amsterdam The netherlands)

14 Storz H and Jelke E (1984) ldquoPhotomicrography of Weakly Fluorescent ObjectsmdashEmployment of p-phenylenediamine as a Blocker of Fading rdquo Acta Histochem 75 133ndash139

OrderCustomer Service (800) 325-3010 bull Fax (800) 325-5052 Technical Service (800) 325-5832 bull sigma-aldrichcomtechservice DevelopmentCustom Manufacturing Inquiries (800) 244-1173 Safety-related Information sigma-aldrichcomsafetycenter

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copy2012 Sigma-Aldrich Co LLC All rights reserved SIGMA and SIGMA-ALDRICH are trademarks belonging to Sigma-Aldrich Co LLC registered in the US and other countries Where bio begins is a trademark of Sigma-Aldrich Co LLC FLAG AnTI-FLAG HIS-Select and ExtrAvidin are registered trademarks of Sigma-Aldrich Co LLC EZ View FLAG-BAP 3xFLAG CelLytic and SIGMAFAST are trademarks of Sigma-Aldrich Co LLC Sepharose is a registered trademark of G E Healthcare TRITOn is a registered trademark of Union Carbide Corp Cy is a trademark of GE Healthcare Benzonase is a registered trademark of Merck KGaA TWEEn is a registered trademark of Unigema a business unit of ICI Americas Inc Sigma brand products are sold through Sigma-Aldrich Inc Purchaser must determine the suitability of the product(s) for their particular use Additional terms and conditions may apply Please see product information on the Sigma-Aldrich website at www sigmaaldrich com andor on the reverse side of the invoice or packing slip