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Poppers™

(easier, faster, safer, gentler)Cell Lysis Solutions

Protein Extraction • Cell Fractionation • DNA Extraction • Protein RefoldingG r a s p t h e P r o t e o m e ™

1For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007

Table 1. Poppers™ Cell Lysis Reagents selection guide.

Popper (Product #) Organisms/Samples Dialyze1 Compatibility Notes

1. The detergent can be removed by dialysis2. Immunoprecipitation3. Halt™ Protease Inhibitor Cocktail, Product # 78410 and 78415 (EDTA-free)4. Samples prepared in Mem-PER® Reagent can be dialyzed if the buffer contains detergent (e.g., CHAPS)5. Slide-A-Lyzer ® MINI Dialysis Units6. 2-D Sample Prep for Nuclear Proteins (Product # 89863) and 2-D Sample Prep for Membrane Proteins

(Product # 89864) are designed using our popular NE-PER ® and Mem-PER ® Reagents.

† B-PER® Technology is protected by U.S. patent # 6,174,704. U.S. patent pending on Mitochondria Isolation Kit Technology.

Protease inhibitors may be added to prevent proteindegradation. Douncing will increase isolation efficiency vs. detergent alone; however, multiple samples can be processed simultaneously using the reagent-based methods.

Western blot, 2-D Western blots, electrophoresis. Applications includeapoptosis, signal transduction andmetabolic studies.

NAMammalian cells

Heart, liver, kidney and brain

Mitochondria IsolationKit for Cultured Cells†

89874Mitochondria IsolationKit for Tissue†

89801

Protease inhibitors3 may be added to prevent proteindegradation. Can dialyze against another detergent(e.g., CHAPS). Extraction efficiency is generally > 50%with the cell lines tested (having proteins with up totwo transmembrane segments).

Western blot and 2-D6Yes4Cultured cells: brain (C6), epithelial(HeLa), fibroblasts (NIH3T3), yeast (S. cerevisiae)

Mem-PER® Reagent89826

Protease inhibitors3 may be added to prevent proteindegradation. Packed cell vol.: 2 x 106 HeLa cells = 10 µl= 20 mg. Tissue yield (calf liver): 3-4 mg cytoplasmicprotein/100 mg tissue; 1-1.5 mg nuclear protein/100 mgtissue. Cell yield (HeLa): 300-400 µg cytoplasmicprotein/106 cells; 40-60 µg nuclear protein/106 cells.Positive controls tested: cytoplasmic (β-Gal, PKC,Hsp90); nuclear (Oct-1, p53, DNA polymerase)

EMSA (if using < 3 µl or 10%, otherwise dialyze first in SAL MINIs5),Western blot, reporter assays, IEF (after dialysis to reduce salt con-centration) and 2-D6

No(CER)Yes

(NER)

Tissue: calf liver. Cultured cells: epithelial (HeLa),fibroid (COS-7), kidney (NIH3T3), liver (Hepa 1), brain (C6)

NE-PER® Reagent78833

Protease inhibitors3 may be added to prevent proteindegradation. Mechanical disruption of the tissue isstill required. Can also be used for cultured cells.

Luciferase, β-Gal, CAT, kinase assays,Western blot, ELISAs, immobilized glutathione

YesHeart, liver, kidney and brainT-PER® Reagent78510, 500 ml

Protease inhibitors3 may be added to prevent proteindegradation. Adding 150 mM NaCl results inincreased efficiency of lysis and higher protein yieldin some cells lines. A PBS rinse of cells prior to lysis removes contaminants such as phenol redand increases protein yield.

Luciferase, β-Gal (low signal), CAT,kinase assays, ELISAs, immobilized glutathione, Western blot

YesCultured mammalian cells, COS-7,NIH3T3, Hepa 1-6, 293, CHO, MDA,MB231, FM2 and insect cells

M-PER® Reagent78503, 25 ml78501, 250 ml78505, 1 L

Protease inhibitors3 may be added to prevent proteindegradation. The addition of up to 2 M NaCl may resultin increased efficiency of lysis and protein yield.

GST- and histidine-tag purification, Western blot

YesYeast (S. cerevisiae),Acinetobacter sp.

Y-PER® Plus Reagent78998, 25 ml78999, 500 ml

Protease inhibitors3 may be added to prevent proteindegradation. Use at room temperature. Double incu-bation time for use at 4°C. Use log phase cells. Forstationary phase cells, add 0.1 M DTT or 20-50 mMTCEP. Will work with 1 mM EDTA. Does not lysespores. Cannot use with ion exchange columns.

IPs2, Western blot, β-Gal enzymeassays, IEF after dialysis, GST- and histidine-tag purification

NoS. cerevisiae, Schizo-saccharomycespombe, C. albicans, B. subtilis, E. coli, P. pastoris, Strep. avidinii,Acinetobacter sp.

Y-PER® Reagent75768, 25 ml78991, 200 ml78990, 500 ml

Protease inhibitors3 may be added to prevent proteindegradation. Salts, chelating agents and reducingagents can be added for more efficient lysis. Betterlysis if cells are frozen in B-PER® Reagent.

Reporter assays, IPs2, Western blot,GST- and histidine-tag purification

YesGram(-) bacteria, S. aureus, H. pylori,E. coli strains BL21(D3)> JM109>DH5α>M15, Archaebacteria, nema-todes, Acinetobacter sp., Insect cells

B-PER® PBS Reagent78266, 500 ml

Protease inhibitors3 may be added to prevent proteindegradation. Salts, chelating agents and reducingagents can be added for more efficient lysis. Betterlysis if cells are frozen in B-PER® Reagent.


YesGram(-) bacteria, S. aureus, H. pylori, E. coli strains- BL21(D3)> JM109>DH5α>M15, Archaebacteria, nema-todes, Acinetobacter sp., Insect cells

B-PER® II Reagent78260, 250 ml(A 2X version of B-PER® Reagent)

Protease inhibitors3 may be added to prevent proteindegradation. Salts, chelating agents and reducingagents can be added for more efficient lysis. Do notexceed 0.5 M NaCl. Better lysis if cells are frozen inB-PER® Reagent.


YesGram(-) bacteria, S. aureus, H. pylori, E. coli strains BL21(D3)> JM109>DH5a >M15, Archaebacteria, nema-todes, Acinetobacter sp., insect cells

B-PER® Reagent†

78243, 165 ml78248, 500 ml

Contents

www.piercenet.com/pop95d • E-mail Customer Service: [email protected]

(easier, faster, safer, gentler)Cell Lysis Solutions

Poppers

Poppers™ Cell Lysis Reagents Selection Guide...........................................1

Introduction to Protein Extraction.........................................................2-4Cell Lysis Methods....................................................................................................3-4

Introduction to Poppers™ Cell Lysis Solutions ........................................5-10B-PER® Bacterial Protein Extraction Reagents..........................................................6-7M-PER® Mammalian Protein Extraction Reagent .........................................................8T-PER® Tissue Protein Extraction Reagents .................................................................9Y-PER® Yeast Protein Extraction Reagents .............................................................9-10

Fusion Protein Purification .............................................................11-16Fusion Protein Purification Kits ............................................................................11-16GST Orientation Kit ....................................................................................................16

Mammalian and Yeast β-Gal Kits .....................................................17-18

Cell Fractionation ........................................................................19-25Cell Fractionation Introduction ...................................................................................19Mem-PER® Eukaryotic Membrane Protein Extraction Kit......................................20-21Mitochondria Isolation Kits...................................................................................22-23NE-PER® Nuclear and Cytoplasmic Extraction Kit.................................................24-25

DNA Extraction................................................................................26Y-DER® Yeast DNA Extraction Kit ...............................................................................26Lyse-N-Go™ PCR Reagent ..........................................................................................26

Detergents.................................................................................27-31Introduction to Detergents .........................................................................................27Properties of Common Detergents.............................................................................29Surfact-Amps® and Surfact-Pak™ Detergents.............................................................29Specialized Detergents..........................................................................................30-31

Protease Inhibitors...........................................................................32Introduction to Protease Inhibitors ............................................................................32Halt™ Protease Inhibitor Cocktails ..............................................................................32PMSF .........................................................................................................................32Soybean Trypsin Inhibitor ..........................................................................................32

Protein Refolding.........................................................................33-35Pro-Matrix™ Protein Refolding Kit ..............................................................................33Inclusion Body Solubilization Reagents .....................................................................34

Protein Desalting.............................................................................35Zeba™ Desalt Spin Columns .......................................................................................35

Poppers™

Cell Lysis Solutions

Introduction to Protein Extraction

Figure 1. Lipid bilayer constituting the outer plasma membrane of a cell.

Cell Lysis Using Traditional (Non-detergent) Methods

Several methods, including mechanical disruption, liquid homoge-nization, sonication, freeze/thaw cycles and manual grinding (Table 2)are commonly used to physically lyse cells. These methods havebeen reviewed extensively.1-4

Mechanical DisruptionMechanical methods rely on the use of rotating blades to grindand disperse large amounts of complex tissue, such as liveror muscle. The Waring® Blender and the Polytron® Mixer arecommonly used for this purpose. Unlike the Waring® Blender,which is similar to a standard household blender, the Polytron®

Mixer draws tissue into a long shaft containing rotating blades.The shafts vary in size to accommodate a wide range of volumes,and can be used with samples as small as 1 ml.

Liquid HomogenizationLiquid-based homogenization is the most widely used cell-disruptiontechnique for small volumes and cultured cells. Cells are lysed byforcing the cell or tissue suspension through a narrow space,thereby shearing the cell membranes. Three different types ofhomogenizers are in common use. A Dounce homogenizer con-sists of a round glass pestle that is manually driven into a glasstube. A Potter-Elvehjem homogenizer consists of a manually ormechanically driven Teflon® Coated Pestle shaped to fit a roundedor conical vessel. The number of strokes and the speed at whichthe strokes are administered influences the effectiveness ofDounce and Potter-Elvehjem homogenization methods. Bothhomogenizers can be obtained in a variety of sizes to accommodatea range of volumes. A French press consists of a piston that is

used to apply high pressure to a sample volume of 40 to 250 ml,forcing it through a tiny hole in the press. Only two passes arerequired for efficient lysis due to the high pressures used withthis process. The equipment is expensive, but the French press isoften the method of choice for breaking bacterial cells mechanically.

Table 2. Techniques used for the physical disruption of cells.

Lysis Method Apparatus DescriptionMechanical Waring® Blender Rotating blades grind and

Polytron® Mixer disperse cells and tissuesLiquid Dounce Homogenizer Cell or tissue suspensionsHomogenization Potter-Elvehjem are sheared by forcing them

Homogenizer through a narrow spaceFrench Press

Sonication Sonicator High frequency soundwaves shear cells

Freeze/Thaw Freezer or dry ice/ Repeated cycles of freezing ethanol and thawing disrupt cells

through ice crystal formationManual Grinding Mortar and pestle Grinding plant tissue, frozen

in liquid nitrogen

SonicationSonication is the third class of physical disruption commonlyused to break open cells. The method uses pulsed, high-frequencysound waves to agitate and lyse cells, bacteria, spores, and finelydiced tissue. The sound waves are delivered using an apparatuswith a vibrating probe that is immersed in the liquid cell suspension.Mechanical energy from the probe initiates the formation ofmicroscopic vapor bubbles that form momentarily and implode,causing shock waves to radiate through a sample. To preventexcessive heating, ultrasonic treatment is applied in multipleshort bursts to a sample immersed in an ice bath. Sonication isbest suited for volumes < 100 ml.

Freeze/Thaw The freeze/thaw method is commonly used to lyse bacterial andmammalian cells. The technique involves freezing a cell suspensionin a dry ice/ethanol bath or freezer and then thawing the materialat room temperature or 37°C. This method of lysis causes cellsto swell and ultimately break as ice crystals form during the freezingprocess and then contract during thawing. Multiple cycles arenecessary for efficient lysis, and the process can be quite lengthy.However, freeze/thaw has been shown to effectively releaserecombinant proteins located in the cytoplasm of bacteria3 and isrecommended for the lysis of mammalian cells in some protocols.4

Protein purification encompasses total protein extraction

from a sample (lysis), specific enrichment and/or isolation

of a particular protein of interest (affinity purification), and

removal of interfering or contaminating substances (sample

preparation or clean-up).

Cell lysis is the first step in cell fractionation and protein

purification and, as such, opens the door to a myriad of

biological studies. Many techniques are available for the

disruption of cells, including physical and detergent-

based methods. Historically, physical lysis has been the

method of choice for cell disruption; however, physical

methods often require expensive, cumbersome equipment

and involve protocols that can be difficult to repeat

due to variability in the apparatus (such as loose-fitting

compared with tight-fitting homogenization pestles). In

recent years, detergent-based lysis has become very

popular due to ease of use, low cost and efficient

protocols. Pierce offers several detergent-based Poppers™

Reagents for the preparation of whole and fractionated

cell lysates that are faster and more convenient than

traditional lysis methods.

All cells have a plasma membrane, a protein-lipid bilayer that formsa barrier separating cell contents from the extracellular environment.Lipids constituting the plasma membrane are amphipathic, havinghydrophilic and hydrophobic moieties that associate spontaneouslyto form a closed bimolecular sheet (Figure 1). Membrane proteinsare embedded in the lipid bilayer, held in place by one or moredomains spanning the hydrophobic core. In addition, peripheralproteins bind the inner or outer surface of the bilayer through interac-tions with integral membrane proteins or with polar lipid head groups.The nature of the lipid and protein content varies with cell type.

In animal cells, the plasma membrane is the only barrier separatingcell contents from the environment, but in plants and bacteria theplasma membrane is also surrounded by a rigid cell wall. Bacterialcell walls are composed of peptidoglycan. Yeast cell walls arecomposed of two layers of β-glucan, the inner layer being insolubleto alkaline conditions. Both of these are surrounded by an outerglycoprotein layer rich in the carbohydrate mannan. Plant cell wallsconsist of multiple layers of cellulose. These types of extracellularbarrier confer shape and rigidity to the cells. Plant cell walls areparticularly strong, making them very difficult to disrupt mechanicallyor chemically. Until recently, efficient lysis of yeast cells requiredmechanical disruption using glass beads. Bacterial cell walls arethe easiest to break compared to these other cell types. The lack ofan extracellular wall in animal cells makes them relatively easy to lyse.

Clearly, the technique chosen for the disruption of cells, whetherphysical or detergent-based, must take into consideration the originof the cells or tissues being examined and the inherent ease ordifficulty in disrupting their outer layer(s). In addition, the methodmust be compatible with the amount of material to be processedand the intended downstream applications. This handbook discussesboth non-detergent and detergent-based lysis techniques and thenintroduces Poppers™ Cell Lysis Solutions.

www.piercenet.com/pop95d • E-mail Customer Service: [email protected] 3For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007

www.piercenet.com/pop95d • E-mail Customer Service: [email protected] 5For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007

Mortar and PestleManual grinding is the most common method used to disruptplant cells. Tissue is frozen in liquid nitrogen and then crushedusing a mortar and pestle. Because of the tensile strength ofthe cellulose and other polysaccharides constituting the cellwall, this method is the fastest and most efficient way to accessplant proteins and DNA.

Additives/FacilitatorsCells can be treated with various agents to aid the disruptionprocess. Lysis can be promoted by suspending cells in ahypotonic buffer, which causes them to swell and burst morereadily by physical shearing. Lysozyme (200 µg/ml) (Product #s89833 and 89834; page 6) can be used to digest the polysac-charide component of yeast and bacterial cell walls. Alternatively,processing can be expedited by treating cells with glass beadsin order to facilitate the crushing of cell walls. This treatment iscommonly used with yeast cells. Viscosity of a sample typicallyincreases during lysis due to the release of nucleic acid material.DNase (Product # 89835; page 6) can be added to samples(25-50 µg/ml) along with RNase (50 µg/ml) to reduce this problem.Nuclease treatment is not required for sonicated materialbecause sonication shears chromosomes. Finally, proteolysis canbe a problem whenever cells are manipulated; therefore, proteaseinhibitors (Halt™ Protease Inhibitors, Product #s 78410 and 78415;page 32) should be added to all samples undergoing lysis.

Cell Lysis Using DetergentsDetergent cell lysis is a milder and easier alternative to phys-ical disruption of cell membranes, although it is often used inconjunction with homogenization and mechanical grindingwith a Polytron® Mixer.

Detergents break the lipid barrier surrounding cells by solubi-lizing proteins and disrupting lipid:lipid, protein:protein andprotein:lipid interactions. Detergents, like lipids, self-associateand bind to hydrophobic surfaces. They are composed of apolar hydrophilic head group and a nonpolar hydrophobic tailand are categorized by the nature of the head group as eitherionic (cationic or anionic), nonionic or zwitterionic. Theirbehavior depends on the properties of the head group and tail.

Unfortunately, there is no standard protocol available forselecting a detergent to use for membrane lysis. The idealdetergent will depend on the intended application. In general,nonionic and zwitterionic detergents are milder and lessdenaturing than ionic detergents and are used to solubilizemembrane proteins when it is critical to maintain proteinfunction and/or retain native protein:protein interactions forenzyme assays or immunoassays. CHAPS, a zwitterionic deter-gent, and the Triton® X Brand series of nonionic detergentsare commonly used for these purposes. In contrast, ionicdetergents are strong solubilizing agents and tend to denatureproteins, thereby destroying protein activity and function. Studiesassessing protein levels strictly through gel electrophoresisand Western blotting typically use SDS to fully denature proteinsamples by boiling. There are a few commonly used ionicdetergents that are only mildly denaturing, including sodiumcholate and sodium deoxycholate.

The choice of detergent for cell lysis also depends on sample type.Animal cells, bacteria and yeast all have differing requirementsfor optimal lysis due to the presence or absence of a cell wall.Because of the dense and complex nature of animal tissues,they require both detergent and mechanical lysis. In addition tothe choice of detergent, other important considerations foroptimal cell lysis include the buffer, pH, salt concentration andtemperature. Consideration should be given to the compatibilityof the chosen detergent with downstream applications. If thedetergent used for lysis must be removed, then a dialyzabledetergent should be selected.

References1. Evans, W.H. (1987). In J.B.C. Findlay and W.H. Evans (Eds.), Biological Membranes:

A Practical Approach. IRL Press, Oxford, England, p. 1-25.2. McNamee, M.G. (1989). Biotechniques 7, 466-4753. Johnson, B.H. and Hecht, M.H. (1994). Bio/Technology 12, 1357-1360.4. Current Protocols in Molecular Biology (1995). John Wiley and Sons, Inc.

(supplement 29) pp. 9.7.1-9.7.2.

To eliminate the need for hit-or-miss homemade recipes, Pierceoffers Poppers™ Cell Lysis Reagents for lysing various sampletypes (Table 1, page 1). Mammalian cells are one of the easiest celltypes to lyse. The M-PER® Mammalian Protein Extraction Reagent(Product # 78501) uses a non-denaturing detergent to preparetotal cell lysate that is compatible with many downstream assaysincluding immunoassays, enzyme assays and a variety of commonreporter assays. The major advantage to the M-PER® Reagent isthat lysis can be performed directly on the plate and is completed inonly 5 minutes. Furthermore, significantly more protein can beobtained with this method compared with freeze/thaw and sonica-tion (Figure 2).

Figure 2. Comparison of M-PER® Reagent with freeze/thaw cycles, sonication andBrand P lysis buffer. COS-7 cells grown in 100 mm plates at full confluencywere washed once with 10 ml of PBS, scraped with 1 ml of PBS and centrifugedat 5,000 rpm for 5 minutes to collect the cells. The cell pellets were resuspendedin 0.5 ml of respective extraction reagents and subjected to total protein extraction.For freeze/thaw cycles, the cell suspension in PBS was frozen in a dry ice andisopropanol bath for 10 minutes and thawed in a 37°C water bath. The freeze/thawcycle was repeated three times. For sonication, the cell suspension was sonicatedfor 2 minutes with a 50% pulse using a Branson Sonifier® 450 Sonicator. Forextraction with M-PER® Reagent and Brand P lysis buffer, the cell suspensions wereshaken for 5 minutes. The cell debris was removed by centrifugation at 13,000rpm for 5 minutes and the supernatants were assayed for protein concentrationby the BCA™ Method.

T-PER® Tissue Protein Extraction Reagent (Product # 78510) isdesigned for total protein extraction from tissue samples. The T-PER® Reagent uses a nondenaturing detergent in 25 mM bicine,150 mM NaCl, pH 7.6 and is used in conjunction with mechanicalor manual homogenization. The resulting cell lysate, like thelysate prepared with M-PER® Reagent, is compatible with manyfunctional assays.

B-PER® Bacterial Protein Extraction Reagent (Product # 78248)is used to gently lyse Escherichia coli and other bacterial cellsand extract soluble recombinant proteins. This simple extractionprocedure does not require expensive equipment and can beperformed in less than 10 minutes. B-PER® Reagent also effec-tively removes soluble protein from inclusion bodies and can beused to purify intact inclusion bodies whose less soluble proteinscan be extracted by other means. B-PER® II Bacterial ProteinExtraction Reagent (Product # 78260) is even more efficient thanthe original B-PER® Reagent in that it extracts more protein anduses smaller volumes.

The tough yeast cell wall can be disrupted without damaging thecell and its contents using Y-PER® Yeast Protein Extraction Reagent(Product # 78990) (Figure 3). The method does not require enzymesor glass beads to aid cell lysis and results in the release of func-tionally active solubilized proteins. The reagent can also be usedeffectively with gram-positive and gram-negative bacteria. Y-PER®

Plus Dialyzable Yeast Protein Extraction Reagent (Product # 78999)is an alternative to the original Y-PER® Reagent. It is phosphate-freeand has a much lower ionic strength than the original Y-PER® Reagent.

The whole cell lysates prepared with all of the above-mentionedPoppers™ Reagents except Y-PER® Reagent are compatible withBCA™ and Coomassie Protein Assays.

Figure 3: Y-PER® Reagent significantly disrupts yeast cell wall and plasmamembrane. Cells of Saccharomyces cerevisiae stain DY150 after lysis with Y-PER®

Yeast Protein Extraction Reagent. Arrows indicate disruption of cell wall, resultingin cell lysis.

M-PER®

ReagentFreeze-Thaw

Cycles

µg P

rote

in

Sonication Brand PLysis Buffer

800

600

400

200

0

For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007

Popp

ers Introduction

Introduction to Poppers™ Cell Lysis Solutions

B-PER® Reagent (in Phosphate Buffer)Yields obtained with B-PER® Reagent (in Phosphate Buffer) exceed those obtained using standard sonication.

Highlights:• Recombinant protein extraction is comparable to that of the

original (Tris buffer-based) B-PER® Bacterial Protein ExtractionReagent formulation

• Does not denature or interfere with recombinant proteins• Will not interfere with amine-reactive chemistries for cross-linking,

biotinylation or immobilization• No need for expensive equipment – complete lysis achieved with

B-PER® Reagent (in Phosphate Buffer)

After recombinant protein is extracted from E. coli, it may be furtherpurified or functionally analyzed. The original B-PER® Reagent usesa Tris buffer (20 mM, pH 7.5) system. For some proteins, however,a phosphate buffer system is preferred. B-PER® Reagent (inPhosphate Buffer) is designed to address this need. The performanceof B-PER® Reagent (in Phosphate Buffer) is comparable to thatof the original (Tris buffer-based) B-PER® Reagent. No significantdifference was observed between these two buffer-based B-PER®

Reagents, indicating that the buffer has no impact on the extractionefficiency (Figure 6).

Figure 6. Comparison of B-PER® Reagent (in Phophate Buffer) with originalB-PER® Reagent. Approximately 1 g of bacterial pellets was extracted five timeswith 10 ml of either B-PER® Reagent (in Phosphate Buffer) or original B-PER®

Reagent. B-PER® Reagent (in Phosphate Buffer) showed a pattern similar to thatof the original B-PER® Reagent; i.e., most of the proteins were extracted in thefirst round. The protein concentration is measured by (A) BCA™ Assay and thegreen fluorescent protein (GFP) activity is determined by a (B) luminescencespectrometer (Perkin-Elmer LS50).

Ordering InformationProduct # Description Pkg. Size78266 B-PER® Reagent (in Phosphate Buffer) 500 ml

Rounds of Extraction

A. B.

µg p

rote

in/m

l

31 2 5

Tota

l

4

Rounds of Extraction31 2 5

Tota

l

4

120

100

80

60

40

20

0

RLU

30,000

25,000

20,000

15,000

10,000

5,000

0

B-PER® (in Phosphate Buffer)B-PER® Reagent

B-PER® (in Phosphate Buffer)B-PER® Reagent

B-PER® II Bacterial Protein Extraction ReagentDeveloped to provide more efficient recombinant protein extraction with smaller volumes than that of the original B-PER® Reagent.

Highlights:• Can be used for soluble protein extraction and inclusion

body purification*• Protein remains concentrated• Uses Tris buffer system• No need for expensive equipment – complete lysis achieved with

B-PER® II Bacterial Protein Extraction Reagent

* Does not solubilize inclusion bodies. See page 34 for information on Pierce InclusionBody Solubilization Reagent.

With small (5 ml) volumes, the yields of both total soluble proteinand recombinant green fluorescent protein (GFP) extracted byB-PER® II Reagent are twice as high as the yields produced byoriginal B-PER® Reagent at the first round (Figure 7). The yieldof both total protein and GFP from all five rounds of extractionare similar. Therefore, B-PER® II Reagent is ideal for recombinantprotein extraction when a small volume is required.

Figure 7. Comparison of B-PER® II Reagent with original B-PER® Reagent.Approximately 1 g of bacterial pellets expressing recombinant GFP was extract-ed five times with 5 ml of either B-PER® II Reagent or original B-PER® Reagent.The protein concentration is measured by BCA™ Assay, and the GFP activity isdetermined by a luminescence spectrometer (Perkin-Elmer LS50). A: solubleproteins extracted in each round. B: recombinant GFP extracted in each round.Note that at the first round of extraction, the yield from B-PER® II Reagent ismuch higher than that of the original B-PER® Reagent.

Ordering InformationProduct # Description Pkg. Size78260 B-PER® II Bacterial Protein Extraction Reagent 250 ml


A. B.

µg p

rote

in/m

l

31 2 5

Tota

l

4Rounds of Extraction

31 2 5

Tota

l

4

120

100

80

60

40

20

0

RLU

30,000

25,000

20,000

15,000

10,000

5,000

0

B-PER® II ReagentB-PER® Reagent

B-PER® II ReagentB-PER® Reagent

B-PER® Bacterial Protein Extraction ReagentYields obtained with B-PER ® Reagent greatly exceed those obtained using standard sonication methods.

Highlights:• Recovers both soluble and insoluble recombinant protein from bacterial

lysates – purifies inclusion bodies to near-homogeneous levels*• One easy-to-use reagent – B-PER® Reagent provides one-step

E. coli cell lysis by a mild, nonionic detergent in 20 mM Tris•HClbuffer (pH 7.5)

• Fast and simple – just add B-PER® Reagent to a bacterial pelletand shake for 10 minutes. Recover soluble proteins by pelletingcell debris. Purify inclusion body* from the pellet using anoptimized procedure

• Flexible – B-PER® Reagent is suitable for any scale protein extraction• Avoids contamination – B-PER® Reagent is free of enzymatic compo-

nents, thus avoiding contamination of your recombinant protein.If necessary, the nonionic detergent can be removed by dialysis

• Compatible with GST, 6xHis and other affinity purifications1

*Does not solubilize inclusion bodies. To solubilize inclusion bodies, see page 34.

The first step to purify or characterize a recombinant protein is todisrupt the cell and release the protein. B-PER® Bacterial ProteinExtraction Reagent offers a gentle method of bacterial cell lysis,while also providing the most efficient way to extract recombinantprotein from E. coli.

Table 3. General considerations for B-PER® Reagent.

Three convenient reagent formats: Original B-PER® Reagent is supplied in20 mM Tris•HCl (pH 7.5), although a phosphate buffer alternative formulationis also available for applications requiring an amine-free buffer. B-PER® IIReagent is a twice-concentrated (2X) version of the original reagent, enablingmore concentrated extracts or addition to cells already in solution.Fresh cells and frozen cells: B-PER® Reagent is capable of extracting proteinsfrom both fresh and frozen cells. However, the extraction is typically mosteffective with frozen cells.E. coli strains: B-PER® Reagents work well for many common bacterial hoststrains. They are especially suitable for the commonly used, protease-defectivebacterial expression host BL21 strains. If the lysis is not efficient for a particularbacterial strain, try freezing the cells before extraction.Soluble proteins and inclusion bodies: Perform a mini-scale extraction todetermine solubility recombinant proteins before performing larger-scaleextractions and purifications. Recombinant proteins expressed in bacteriaoften form inclusion bodies, especially when expressed at high levels. B-PER®

Reagents effectively extract and remove soluble proteins from the insolubleinclusion body pellet. When combined with inclusion body solubilization andprotein refolding, purified recombinant protein may be obtained from theinclusion body pellet.Lysozyme: For inclusion body purification, add lysozyme to further digest celldebris and release the inclusion bodies. Lysozyme digestion can significantlyimprove inclusion body protein purity; the lysozyme will be eliminated duringsubsequent washing steps in the protocol.DNase I: When minimizing the amount of B-PER® Reagent used in an extraction,viscosity caused by DNA may prevent efficient centrifugation of cell debris. Insuch cases, add a small amount of DNase I to clear the solution.Insect cells: B-PER® Reagent has been tested for the extraction of recombinantproteins from insect cells infected by baculovirus. The amount of the reagentrequired depends on the confluency of the infected cells.Optional/supplemental materials: Protease inhibitors, salts, chelating agents,reducing agents, etc. may be added directly to the reagent for specific applications.

** GFP = Green Fluorescent Protein.

Figure 4. Comparison of B-PER® Reagent with sonication. E. coli expressingGFP was extracted five times with B-PER® Reagent or PBS/sonication. Eachextraction was analyzed by SDS-PAGE.

Figure 5. Comparison of B-PER® Reagent with sonication for measurement ofgreen fluorescent protein (GFP). E. coli expressing GFP was extracted fivetimes with B-PER® Reagent or PBS/sonication. Each extraction was analyzed byGFP activity assay.

Reference1. Dorsey, C.W., et al. (2003). Genetic organization of an Acinetobacter baumannii

chromosomal region harbouring genes related to siderophore biosynthesis and trans-port. Microbiology 149, 1227-1238.

Ordering InformationProduct # Description Pkg. Size78248 B-PER® Bacterial Protein Extraction Reagent 500 ml78243 B-PER® Bacterial Protein Extraction Reagent 165 ml89833 Lysozyme 5 g89834 Lysozyme 25 g89835 DNase I 5,000 units


GFP

Activ

ity

31 2 5

Pelle

t4

300

250

200

150

100

50

0

B-PER® ReagentPBS/sonication


Pop

per

s In

trod

uctio

n

For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007 7

Popp

ers Introduction

B-PER® Bacterial ProteinExtr

actio

nRe

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T-PER® Tissue Protein Extraction ReagentDeveloped for extraction of total protein from tissue samples.

Highlights:• Simple procedure – just homogenize tissue sample in 1:20

(w/v) of tissue to T-PER® Reagent, then centrifuge to pelletcell/tissue debris

• Mild detergent is dialyzable for quick and easy removal• Can be used with additional components (e.g., protease

inhibitors, salts, reducing agents, chelating agents, etc.)1-5

• Lysate may be used for reporter assays, protein kinase assays, immunoassays, ELISAs, Western blots and/or protein purifications1-5

• The lysate is compatible with standard protein assays such asPierce Coomassie Plus – The Better Bradford™ Protein Assay(Product # 23236)1-5

This reagent uses a proprietary detergent in 25 mM bicine, 150 mMsodium chloride (pH 7.6) for tissue cell lysis. The simple compositionof this reagent provides versatility for many different applications.Depending on the application, it may be advantageous to add othercomponents such as protease inhibitors, salts, reducing agents,chelating agents, etc., to the reagent before proceeding with the celllysis. The cell lysate prepared with this reagent may be used forreporter assays (e.g., luciferase, β-galactosidase, chloramphenicolacetyl transferase), protein kinase assays (e.g., PKA, PKC, tyrosinekinase), immunoassays (e.g., Western blots, ELISAs, RIAs) and/orprotein purifications.

Protocol1. Weigh tissue samples. A 1:20 (w/v) ratio of tissue to T-PER®

Reagent is optimal for this procedure.Notes:a. Protease inhibitors may be added to the T-PER® Reagent

(if necessary).b. Smaller volumes of T-PER® Reagent may be used if a more

concentrated protein extract is required.2. Add the appropriate amount of T-PER® Reagent to the tissue

sample and homogenize.3. Centrifuge the sample at 10,000 rpm for 5 minutes to pellet

cell/tissue debris.4. Collect supernatant and continue with downstream analysis or

further purification.

References1. Sheng, J.G., et al. (2002). Disruption of corticocortical connections ameliorates amyloid

burden in terminal fields in a transgenic model of Aβ amyloidosis. J. Neurosci. 22(22),9794-9799.

2. Jepsen, K.H., et al. (2002). A syndrome of joint laxity and impaired tendon integrity inlumican- and fibromodulin-deficient mice. J. Biol. Chem. 277, 35532-35540.

3. Runkuan, Y., et al. (2002). Lipopolysaccharide induces overexpression of MUC2 andMUC5AC in cultured bililary epithelial cells: possible key phenomenon of heptatolithiasis.Amer. J. Pathol. 161, 1475-1484.

4. Lukashevich, I.S., et al. (2003). Arenavirus-mediated liver pathology: acute lymphocyticchoriomeningitis virus infection of rhesus macaques is characterized by high-levelinterleukin-6 expression and hepatocyte proliferation. J. Virol. 77(3), 1727-1737.

5. Aldred, M.A., et al. (2003). Caveolin-1 and caveolin-2, together with three bone morpho-genetic protein-related genes, man encode novel tumor suppressors down-regulated insporadic follicular thyroid carcinogenesis. Cancer Res. 63, 2864-2871.

Ordering InformationProduct # Description Pkg. Size78510 T-PER® Tissue Protein Extraction Reagent 500 ml89833 Lysozyme 5 g89834 Lysozyme 25 g89835 DNase I 5,000 units78410 Halt™ Protease Inhibitor Cocktail Kit Kit

Sufficient reagents for 200 ml of extract.78415 Halt™ Protease Inhibitor Cocktail, EDTA-Free 1 ml

Sufficient reagents for 100 ml of extract.

Y-PER®-Plus Dialyzable Yeast Protein Extraction Reagent

Highlights:• Entire formulation (including the detergent) is dialyzable• Fast – lysis complete after a 20-minute hands-off incubation• No reducing agents or chelating agents• No rigorous mechanical or lengthy enzymatic treatments • Buffer formulation has a very low (< 100 mM) ionic strength • Buffer formulation does not include NaCl• Tris-based buffer, not phosphate-based (some applications do

not work as well with phosphate-based buffer)• Compatible with both the BCA™ and Coomassie Plus Protein Assays• May be stored at room temperature

Use Y-PER®-Plus Reagent to extract functional soluble proteinsand proteins from S. cerevisiae and P. pastoris (yeast), B. subtilis(Gram-positive bacteria), and E. coli (Gram-negative bacteria).Protein extraction is achieved by a 20-minute incubation in thereagent at room temperature.

Ordering InformationProduct # Description Pkg. Size78998 Y-PER®-Plus Dialyzable Yeast Protein 25 ml

Extraction Reagent78999 Y-PER®-Plus Dialyzable Yeast Protein 500 ml

Extraction Reagent

M-PER® Mammalian Protein Extraction ReagentDesigned to provide highly efficient total protein extraction from cultured mammalian cells.

Highlights:• Mild detergent lysis, yielding extracts that are immediately compatible

with Coomassie (Bradford) and BCA™ Protein Assays or SDS-PAGE1

• Extracts soluble proteins in nondenatured state, enabling direct usein immunoprecipitation and other affinity purification procedures

• Amine-free and fully dialyzable formulation ensures compatibilitywith subsequent assay systems

• Lyse adherent cells directly in plate or after scraping and wash-ing in suspension

• Maintain luciferase, β-Galactosidase, CAT and other reportergene activities as well or better than competitor products andfreeze/thaw methods

To achieve complete cell lysis a unique detergent was chosen thatdissolves cell membranes at low concentrations, does not denatureprotein and is compatible with downstream assays. M-PER®

Reagent extracts 25% and 20% more protein than freeze/thawcycles and sonication, respectively (Figure 2, page 5).

M-PER® Reagent is suitable for cell lysis on all sizes of culture platesLysis with M-PER® Reagent is so efficient that adherent cells donot need to be scraped from the culture dish, especially importantwhen the cells are grown in small-welled plates, such as 96- or24-well plates.

Total protein was recovered efficiently without scraping the cellsby simply adding an appropriate amount of M-PER® Reagent andshaking for 5 minutes as compared with the Brand P lysis buffer in100 mm, 60 mm, 6-well, 24-well and 96-well plates. (Data availableon the Pierce web site.)This feature also provides the feasibility forhigh-throughput cell lysis and subsequent screening assays.

M-PER® Reagent is compatible with reporter assays, kinaseassays, immunoassays and protein assaysM-PER® Reagent is compatible with (A) luciferase, (B) β-Galactosidaseand (C) CAT assays, three popular gene regulation reporter assays(Figure 8). Compared to lysing with Brand P lysis buffer followedby one freeze/thaw cycle (as suggested by the manufacturer) orthe standard freeze/thaw method, M-PER® Reagent yielded moreor equivalent enzyme activities.

Figure 8. M-PER® Reagent compatibility with reporter assays in transientlytransfected mammalian cells. Mammalian FM2 cells were transiently transfectedwith a reporter construct containing the luciferase gene. The transfected cells werelysed with either M-PER® Reagent or Brand P lysis buffer and subjected to luciferaseassay. For β-Galactosidase and CAT assays, MDA-MB-231 cells were contransfectedwith reporter constructs expressing β-Galactosidase and CAT, respectively. Thetransfected cells were lysed with M-PER® Reagent or the freeze/thaw method,and the lysates were assayed for β-Galactosidase and CAT activity.

Reference1. Banyard, J., et al. (2003). Type XXIII collagen, a new transmembrane collagen identified

in metastatic tumor cells. J. Biol. Chem. 278(23), 20989-20994.

Ordering InformationProduct # Description Pkg. Size78503 M-PER® Mammalian Protein Extraction Reagent 25 ml78501 M-PER® Mammalian Protein Extraction Reagent 250 ml78505 M-PER® Mammalian Protein Extraction Reagent 1 L

M-PER® Reagent Brand P + Freeze/Thaw Cycles Freeze/Thaw Cycles

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Y-PER® Yeast Protein Extraction ReagentEasy-to-use solution gently disrupts the tough yeast cell wall in less than 20 minutes at room temperature.

Highlights:• Extracts more than twice as much protein as glass bead methods

(Figure 9)• Eliminates the physical problems associated with traditional glass

bead lysis (e.g., clinging static-charged beads, protein/beadclumps and runaway beads)

• Works with Saccharomyces cerevisiae, Schizosaccharomycespombe, Pichia pastoris and Bacillus subtilus

• Effective for many different organisms, including gram-positiveand gram-negative bacteria (Figure 10); suitable for use in adiverse range of situations

Traditionally, protein extraction from yeast required physical dis-ruption to break through the thick proteinaceous cell envelope;less disruptive lysis methods were possible only with otherorganisms like E. coli. Y-PER® Yeast Protein Extraction Reagentwas the first commercially available yeast lysis reagent to use amild detergent lysis procedure to efficiently release functionallyactive solubilized proteins. Several uses for the Y-PER® Reagenthave been optimized that encompass a broad array of applicationsranging from fusion-tagged protein purification to microplate-compatible enzyme assays, and genomic and plasmid DNA extractionfrom yeast. Y-PER® Reagent has even been used to isolate yeastkiller virus double-stranded RNA from killer strains of S. cerevisiae.1

ProtocolThe standard protocol for protein extraction is easy: 1. Add an appropriate volume of Y-PER® Reagent to pelleted

yeast cells.2. Incubate at room temperature for approximately 20 minutes.3. Spin down the debris.The resulting supernatant is a concentrated protein solution, surpassingtypical yields obtained with traditional glass bead disruption.

Figure 9. Y-PER® Reagent extraction yields greater amounts of usable protein.In all three organisms tested, Y-PER® Reagent extracts contain more usable pro-tein than traditional glass bead lysis.

Figure 10. Y-PER® Yeast Protein Extraction Reagent. Y-PER® Reagent extractionof protein from two different strains each of S. cerevisiae, S. pombe, B. subtilisand E. coli.

Reference1. Liermann, R.T., et al. (2000). BioTechniques 28, 64-65.

Ordering InformationProduct # Description Pkg. Size78990 Y-PER® Yeast Protein Extraction Reagent 500 ml78991 Y-PER® Yeast Protein Extraction Reagent 200 ml

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Fusion Protein Purification Kits

Highlights of both kits:• Greater convenience – no sonication required;

complete cell lysis achieved with B-PER® Reagent• Ready-to-use components

Highlights of the B-PER® 6xHis Fusion Protein ColumnPurification Kit:• Optimized system provides the best purity in the least

amount of time (works in 2.5-3 hours)• High capacity – kit makes it possible to purify more than

10 mg of over-expressed protein per column

Highlights of the B-PER® 6xHis Fusion Protein Spin Purification Kit:• Works in less than 30 minutes• Includes an optimized protocol that makes it possible to achieve

yields approximately 1 mg of pure 6xHis-tagged fusion protein• Includes Handee™ Spin Columns and Collection Tubes

The B-PER® 6xHis Column and Spin Purification Kits are designedfor rapid and efficient purification of 6xHistidine-tagged fusionproteins from bacteria. These kits have also been used for thepurification of 6xHistidine-tagged proteins from baculovirus-infectedinsect cells. As the name suggests, six consecutive histidine residuesare recombinantly fused to the protein of interest and expressed inbacteria. The resulting 6xHis fusion protein can be extracted frombacteria by using B-PER® Bacterial Protein Extraction Reagent andthen purified using one of the pre-packaged Nickel Chelated Columns(Ni-Chelated Columns) or Handee™ Spin Columns included. Thepatented detergent in B-PER® Reagent, combined with a smallamount of imidazole, efficiently washes off nonspecifically and/orweakly bound proteins (e.g., proteins rich in histidine residues).The 6xHistidine-tagged proteins are then eluted with excess imida-zole (Elution Buffer).

The B-PER® 6xHis Fusion Protein Column KitThe kit protocol has been optimized to give a high yield of pure6xHistidine fusion protein in the fastest amount of time. All of thebuffers have been optimized for the most efficient purification. Useof other buffer formulations may significantly alter not only thebinding efficiency of the column, but also the yield and/or the purityof the 6xHistidine-tagged protein. The column has been tested forloading up to 20 ml lysates from 500 ml cultures. However, for optimalresults, a 10 ml lysate from 250 ml bacterial culture (O.D.600 ~1.5-3)is suggested as the starting material. Loading a sample size overthe column capacity may cause the proteins to precipitate on thecolumn and thus stop or hinder the flow-through. The yield andpurity greatly depend on the expression level and the nature of therecombinant protein. As an example, we routinely obtain 10-12 mgof 6xHistidine-tagged green fluorescent protein (GFP) from 250 mlovernight bacterial culture with more than 90% purity using theprotocol found in the kit instructions.

B-PER® Column Kit (Product # 78100) ExampleUsing 6xHis-tagged GFP as amodel, the purification began with the extraction of the recombinantprotein from bacterial cell pellets using B-PER® Reagent. Typically, bacterial cell pellets from 250 ml cultureswere resuspended in 10 ml of B-PER® Reagent. The resuspendedcells were shaken at room temperature (RT) for 10 minutes toensure complete cell lysis and maximal soluble protein extraction.The lysate was centrifuged and the supernatant, which containedthe soluble proteins, was loaded onto a Ni-chelated column. Thecolumn was washed twice with 3 ml of 6xHis Wash Buffer 1 andthree times with 3 ml of 6xHis Wash Buffer 2. The 6xHis WashBuffer 1 and 6xHis Wash Buffer 2 efficiently remove nonspecificproteins from the Ni-chelated column.

The 6xHis-tagged GFP was eluted from the column with the 6xHisElution Buffer (200 mM imidazole). Using this optimized system,12 mg of recombinant GFP was purified from 250 ml of bacterialculture within 3 hours (Figure 11).

M 1 2 3 4 5 6 7 8 9

Figure 11. SDS-PAGE analysis of the purification of 6xHis-tagged GFP fromE. coli using the B-PER® 6xHis Fusion Protein Column Purification Kit.Fractions were collected from each of the purification steps as described in thetext and assayed on a gradient 4%-20% SDS-PAGE gel. The gel was stainedwith GelCode™ Blue Stain Reagent (Product # 24592). Lane 1. crude lysateextracted from E. coli expressing 6xHis-tagged GFP using B-PER® Reagent, Lane 2.flow-through of the lysate after loading onto a Ni-chelated column, Lanes 3-4.two washes with 3 ml of 6xHis Wash Buffer 1, Lanes 5-7. three washes with3 ml of 6xHis Wash Buffer 2, and Lanes 8-9. 6xHis-tagged GFP eluted from thecolumn with 6xHis Elution Buffer. Lane M is the molecular weight marker.

Continues on next page.


B-PER ® 6xHis Fusion Protein Column

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Optimized high-capacity purifications.




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B-PER® Spin Kit (Product # 78300) ExampleUsing 6xHis-tagged GFP as a model, purification begins withrecombinant protein extraction from bacterial cell pellets via B-PER®

Reagent. Bacterial pellets expressing 6xHis-tagged GFP from 250 mlcultures were resuspended in 10 ml of B-PER® Reagent and shakenfor 10 minutes at room temperature (RT) to ensure complete celllysis and maximal soluble protein extraction. The cellular debris wasremoved by centrifugation and the supernatant, which contained6xHis-tagged GFP, was incubated with 1 ml (50% bed resin) ofnickel-charged resin for 10 minutes with gentle shaking. Aftercollecting the affinity gel by a brief, low-speed centrifugation, thegel was resuspended in 0.25 ml of 6xHis Wash Buffer and transferredto a spin column (0.75 ml per column). The resin was washed oncewith 0.5 ml of 6xHis Wash Buffer to remove contamination and therecombinant 6xHis GFP was eluted four times with 6xHis ElutionBuffer (Figure 12). From loading the gel into the column to elutingrecombinant protein, the entire process takes less than 30 minutes.The yield and purity of 6xHis GFP from each elution are shown inTable 4.

Table 4. The yield and purity of four eluents of 6xHis GFP.

Elution 1 2 3 4Yield (mg) 1.6 0.8 0.25 0.1Purity (%) 80.7 90.0 95.1 97.8

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Figure 12. Purification of 6xHis-tagged GFP using the B-PER® Fusion ProteinSpin Kit. Recombinant 6xHis-tagged GFP expressed in E. coli BL21 was firstextracted by B-PER® Reagent (Lane 1). After binding to affinity gel (Lane 2), the6xHis-tagged GFP-bound gels were transferred to spin columns and washedonce with wash buffer to remove contamination (Lane 3). The recombinant pro-teins were eluted four times to achieve complete elution. Lanes 4-5 are eluent 2and eluent 3 of 6xHis-tagged GFP. Lane M is the molecular weight marker.

Ordering InformationProduct # Description Pkg. Size78100 B-PER® 6xHis Fusion Protein Column Purification Kit Kit

Sufficient reagents for five 6xHis purifications (250 ml per culture).Includes: B-PER® Bacterial Protein Extraction Reagent 165 ml

6xHis Wash Buffer 1 45 ml6xHis Wash Buffer 2 60 mlElution Buffer 45 mlNickel Chelated Column 5 x 1 ml

78300 B-PER® 6xHis Fusion Protein Spin Purification Kit KitIncludes: B-PER® Bacterial Protein Extraction Reagent 165 ml

6xHis Wash Buffer 40 mlElution Buffer 45 mlNickel Chelated Agarose 8 mlHandee™ Spin Columns 16 eachCollection Tubes 80 each

89833 Lysozyme 5 g89834 Lysozyme 25 g89835 DNase I 5,000 units78410 Halt™ Protease Inhibitor Cocktail Mix Kit78415 Halt™ Protease Inhibitor Cocktail Mix, 1 ml

EDTA-Free

B-PER® GST Fusion Protein Column and Spin Purification KitsTwo convenient kits that purify > 10 mg/column or milligram quantities in 30 minutes.

Highlights of both kits:• Greater convenience – no sonication required; complete cell lysis

achieved with B-PER® Reagent• Ready-to-use components

Highlights of the B-PER® GST Fusion Protein ColumnPurification Kit:• Fast – from lysis to purified protein in less than two hours• High yields – obtain four times as much protein as other

commercially available kits

Highlights of the B-PER® GST Fusion Protein Spin Purification Kit:• Works in less than 30 minutes• Includes an optimized protocol that makes it possible to achieve

yields approximately 1 mg of pure 6xHis-tagged fusion protein• Includes Handee™ Spin Columns and Collection Tubes

The B-PER® GST Fusion Protein Column and Spin Purification Kitsare designed for rapid purification of glutathione S-transferase (GST)fusion protein from bacteria. The recombinantly expressed GSTfusion protein can be extracted from bacteria by using the B-PER®

Bacterial Protein Extraction Reagent, and then purified usingImmobilized Glutathione included in each kit. The proprietarydetergent in B-PER® Reagent prevents most of the non-GST proteinfrom binding to the column and efficiently washes off nonspecificallybound proteins, providing a rapid and efficient tool for GST fusionprotein purification.

B-PER® Column Kit (Product # 78200) ExampleThe protocol has been optimized to give a high yield of pure GSTfusion protein in the fastest amount of time. All of the buffers havebeen optimized for the most efficient purification. Use of other bufferformulations may significantly alter not only the binding efficiencyof the column, but also the volumes of wash buffer needed to ensurea good purification. For optimal recovery, a sample size should bechosen such that the expected GST load on the column is 80% ofthe maximum (capacity approximately 8 mg GST/column). A 10 mllysate from 250 ml bacterial culture (O.D.600 ~1.5-3) is suggested asthe starting material. Loading a sample size over column capacitymay cause the proteins to precipitate on the column and thus stopor hinder the flow-through.

The B-PER® GST Fusion Protein Column and Spin Purification Kitsuse B-PER® Reagent as the key component. B-PER® Reagent was firstused to extract soluble proteins from Escherichia coli expressingGST. The extracted lysates were then loaded onto an immobilizedglutathione column. The GST protein bound efficiently to theimmobilized glutathione affinity matrix, while other proteins wereprevented by the B-PER® Reagent from binding to the affinity matrix.Nonspecifically bound proteins were further washed away withWash Buffer 1, which contains 50% B-PER® Reagent. Wash Buffer2, which does not contain B-PER® Reagent, was used to equili-brate the column before the elution of the GST protein from thecolumn. As shown in Figure 13, using the B-PER® GST Column

Purification Kit, 5 mg of GST protein was purified from 250 ml ofbacterial culture in 2.5 hours. In comparison with a leading com-petitor’s kit (Figure 14), the B-PER® GST Column Purification Kitproduced four times greater yield without compromising the purity.The higher yield is mainly due to the higher efficiency of solubleprotein extraction from E. coli.

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Figure 13. SDS-PAGE analysis of the GST purification using B-PER® GSTFusion Protein Column Purification Kit. Fractions from each purification stepwere subjected to SDS-PAGE analysis using a gradient 4%-20% SDS-PAGE geland stained with GelCode™ Blue Stain Reagent. Lane 1. the crude lysate extractedfrom E. coli with B-PER® Reagent, Lane 2. the flow-through from the crudelysate, Lanes 3-4. wash fractions of Wash Buffer 1, Lane 5. wash fractions ofWash Buffer 2, and Lane 6: GST eluted from the column with the elution buffer(50 mM glutathione).

Figure 14. Comparison of the B-PER® GST Fusion Protein Column PurificationKit vs. a similar kit from a leading competitor (Brand P). The pure protein yieldfrom the B-PER® GST Kit is approximately four times higher than that ofBrand P. The “W” represents the wash steps and the “E” represents the elution.Note that most of the GST comes off in the first elution with the B-PER® GST Kit,while the majority of the GST protein is found in the second elution fraction withthe competitor’s kit.


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B-PER® 6xHis Fusion Protein Column and Spin Purification Kits (cont.)

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B-PER® GST Fusion Protein Column and Spin Purification Kits (cont.)

B-PER® Spin Kit (Product # 78400) ExampleIn Figure 15, the bacterial extracts containing GST were incubatedwith 1.0 ml of immobilized glutathione (50% resin) agarose for10 minutes with gentle shaking. Following centrifugation, thesupernatant was discarded and the resin containing bound GSTwas resuspended in 0.25 ml of GST Wash Buffer. The resuspendedresin was then transferred to a spin column (0.75 ml per spin column)and placed in 2 ml collection tubes. Following a brief spin, the GSTprotein was eluted four times with 0.5 ml of 25 mM glutathione(reduced) to achieve complete elution. The GST yield and purityare listed in Table 5.

Table 5. The yield and purity of four eluents of GST GFP.

Elution 1 2 3 4Yield (mg) 1.5 0.7 0.3 0.1Purity (%) 92.0 95.1 97.0 99.1

The development of B-PER® Reagent has simplified the extractionof recombinant proteins. Microspin columns provide speed, convenience and flexibility for protein research. The B-PER® GSTFusion Protein Spin Purification Kit provides a highly efficientsystem for GST fusion protein purification.

M 1 2 3 4 5

Figure 15. Purification of GST-tagged GFP using the B-PER® Spin Kit.Recombinant GST-tagged GFP expressed in E. coli BL21 was first extracted byB-PER® Reagent (Lane 1). After binding to affinity gel (Lane 2), the GST-taggedGFP-bound gel was transferred to spin columns and washed once with washbuffer to remove contamination (Lane 3). The recombinant proteins were elutedfour times to achieve complete elution. Lanes 4-5 are eluent 2 and eluent 3 ofGST-tagged GFP.

Ordering InformationProduct # Description Pkg. Size78200 B-PER® GST Fusion Protein Column Purification Kit Kit

Sufficient reagents for five GST fusion protein purifications (250 ml per culture).Includes: B-PER® Bacterial Protein Extraction Reagent 165 ml

Immobilized Glutathione Columns 5 x 1 mlWash Buffer 1 60 mlWash Buffer 2 85 mlGlutathione 1 gm

78400 B-PER® GST Fusion Protein Spin Purification Kit KitIncludes: B-PER® Bacterial Protein Extraction Reagent 165 ml

Immobilized Glutathione Agarose 8 mlWash Buffer 85 mlGlutathione 15 x 16 mgHandee™ Spin Columns 16 eachCollection Tubes 80 each

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Highlights of both kits:• Provides a high yield of purified protein in a short amount of time• Buffers are optimized for the most efficient purification• Purify 6xHis fusion protein from Saccharomyces cerevisiae,

Schizosaccharomyces pombe and Bacillus subtilis

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Figure 16. Purification of a 6xHis-tagged protein from S. cerevisiae using Y-PER®

Reagent. Strain DY150 carrying plasmid pYIL042C was grown and inducedwith galactose to produce a 6xHis-tagged protein. Induced cells were harvestedat an OD600 of 5.3 and lysed using Y-PER® Reagent for 20 minutes. Extract wasapplied to a nickel-chelated agarose column and, after washing, purified 6xHis-tagged Protein (approx. 48 kDa) was eluted using imidazole. Lane 1. Lysate,Lane 2. Column flow-through, Lane 3. Wash 1, Lane 4. Wash 2, Lane 5. Elutionfraction 1, Lane 6. Elution fraction 2, Lane 7. Elution fraction 3, Lane 8. Elutionfraction 4, Lane 9. Elution fraction 5 and Lane 10. Elution fraction 6.

Eukaryotic microorganisms have repeatedly proven their fundamentalworth in studies relating to functional genomics and drug discovery.The unicellular nature of yeast, combined with its ability to performeukaryotic post-translational modifications that closely mimic pro-cesses in higher eukaryotes, has made them important researchtools. The many vectors available, as well as the development oftechniques for working with recombinant expression, have givenyeast an irreplaceable role in the research sector.

Y-PER® 6xHis Column KitThe Y-PER® 6xHis Fusion Protein Column Purification Kit is designedfor rapid and efficient purification of 6xHis fusion proteins fromyeast or bacteria. As the name suggests, six consecutive histidineresidues are fused to the protein of interest as the recombinantprotein is expressed in yeast or bacteria. The resulting 6xHis fusionprotein can be extracted using Y-PER® Yeast Protein ExtractionReagent and subsequently purified using one of the pre-packagednickel chelated columns provided in the kit. The proprietary washbuffers and Y-PER® Reagent efficiently separate nonspecifically and/orweakly bound proteins (e.g., proteins rich in exposed histidine

residues) from the 6xHis fusionprotein that has a strong affinityfor the Ni-chelated column. The6xHis fusion protein is then elutedfrom the column with the application ofa buffer that contains a high concentration of imidazole (Elution Buffer). All contents of this kit are supplied ready to use.

Fresh Cells and Frozen Cells:Y-PER® Reagent is capable of extracting proteins equally well from both freshly harvested and previously frozen cells.

Cell Density and Strain Variation: Differences in organism, media,strain genotype and growth conditions can have dramatic effectson the yield of cells obtained from a given volume of culture.Following are several suggestions for the volume of Y-PER® Reagentto add for a given mass of wet cell paste.

Saccharomyces cerevisiae: Y-PER® Reagent works equallywell on cells grown to saturation or cells isolated from log-phasegrowth in both rich or synthetic defined media. Pierce recommendsusing 2.5-5.0 ml of Y-PER® Reagent for a 1 g cell pellet, whichcan be scaled up or down accordingly.Schizosaccharomyces pombe: Y-PER® Reagent works best oncells grown in media such as Edinburgh Minimal Media (EMM).To achieve adequate lysis of cells grown in rich media such asYES, they must be harvested during log-phase growth. Piercerecommends using 2.5-5.0 ml of Y-PER® Reagent for a 1 g cellpellet, which can be scaled up or down accordingly. Note: For cultures of S. pombe grown past log-phase, increasedincubation temperature (45˚C) and the addition of protease inhibitorsto Y-PER® Reagent has been shown to increase lysis efficiency.Bacillus subtilis: Y-PER® Reagent will not lyse B. subtilis spores.When using a strain that is able to sporulate, take care to harvestthe cells during log-phase growth. For strains unable to sporulate,cells can be grown to saturation prior to lysis. Pierce recommendsusing 2.5-5.0 ml of Y-PER® Reagent for a 1 g cell pellet, whichcan be scaled up or down accordingly.Escherichia coli: Y-PER® Reagent works very well on E. coli.Pierce recommends using 2.5-5.0 ml of Y-PER® Reagent for 1 g cell pellet, which can be scaled up or down accordingly.


B-PER ® 6xHis Fusion Protein Column

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Y-PER® 6xHis and GST Fusion Protein Column Purification KitsHigh-capacity purification > 10 mg/column.

Y-PER® 6xHis and GST Fusion Protein Column Purification Kits (cont.)

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Figure 17. Purification of GST from S. cerevisiae using Y-PER® Reagent. StrainBRS1002 carrying plasmid pYEX4T-2 was grown and induced with copper toproduce GST. Induced cells were harvested in log-phase and lysed usingY-PER® Reagent at room temperature for 20 minutes. Extract was applied toan immobilized glutathione column and, after washing, purified GST (27 kDa)was eluted using 10 mM reduced glutathione. Lane 1. Lysate, Lane 2. Columnflow-through, Lane 3. Wash 1, Lane 4. Wash 2, Lane 5. Elution fraction 1, Lane 6.Elution fraction 2, Lane 7. Elution fraction 3 and Lane 8. Elution fraction 4.

Y-PER® GST Column KitThe Y-PER® GST Fusion Protein Column Purification Kit is designedfor rapid and efficient purification of glutathione S-transferase (GST)fusion proteins from yeast or bacteria. Expressed GST fusion proteinscan be extracted using Y-PER® Yeast Protein Extraction Reagentand subsequently purified using one of the pre-packaged ImmobilizedGlutathione Columns provided in the kit. The proprietary washbuffers and Y-PER® Reagent efficiently separate nonspecificallyand/or weakly bound proteins from the GST fusion protein thathas a strong affinity for the Immobilized Glutathione Column. TheGST fusion protein is then eluted from the column and recoveredin a highly purified form with the application of a buffer that containsa high concentration of reduced glutathione.

Ordering InformationProduct # Description Pkg. Size78994 Y-PER® 6xHis Fusion Protein Column Purification Kit Kit

Contains sufficient reagents for five 6xHis fusion protein purifications from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis or Escherichia coli (up to 6 g of wet cell paste/purification).Includes: Y-PER® Yeast Protein Extraction Reagent 200 ml

Nickel Chelated Columns 5 x 1 mlWash Buffer 1 60 mlWash Buffer 2 60 mlElution Buffer 45 ml

78997 Y-PER® GST Fusion Protein Column Purification Kit KitSufficient reagents for five GST fusion protein purifications from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis or Escherichia coli (up to 6 g of wet cell paste/purification).Includes: Y-PER® Yeast Protein Extraction Reagent 200 ml

Immobilized Glutathione Columns 5 x 1 mlWash Buffer 1 60 mlWash Buffer 2 85 mlGlutathione (reduced) 5 x 184 mg


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GST Orientation KitCovalently attaches purified GST fusion protein to an affinity matrix.

Highlights:• The resulting glutathione support can be used for the

purification of antibody/proteins that have an affinity for thespecific fusion protein

• The purified antibody isolated with the use of this immobilizedantigen support can be used directly for Western blottingapplications, ELISAs and dot blots

Fusion tags are useful tools for the purification of a protein ofinterest. Numerous fusion tags have been developed and, amongthem, the glutathione S-transferase (GST) protein has becomeone of the most widely used tags for protein purification. The GSTOrientation Kit allows the covalent attachment of a GST fusion proteinto glutathione immobilized on a matrix (cross-linked 4% beadedagarose). The resultant affinity matrix can then be used for thepurification of antibodies raised against GST fusion proteins andother ligands that have strong affinity for the GST fusion protein.The purified antibody isolated with the use of this immobilizedantigen support can then be used directly for Western blottingapplications, ELISAs and dot blots.

Figure 18. GST cross-linked to glutathione.

Ordering InformationProduct # Description Pkg. Size78201 GST Orientation Kit Kit

Sufficient reagents to prepare 2 x 2 ml affinity columns, each coupled with 2-15 mg of GST fusion protein and useful for 10 affinity purifications.Includes: Glutathione (reduced) 5 x 184 mg

Immobilized Glutathione 2 x 2 mlBupH™ Modified Dulbecco’s PBS 500 ml

Buffered Saline Pack (Wash Buffer I)ImmunoPure® Elution Buffer 2 x 15 mlImmunoPure® Blocking Buffer 6 mlNeutralization Buffer 5 mlImmunoPure® Gentle Ag/Ab Elution Buffer 100 mlDisuccinimidyl Suberate (DSS) 2 x 13.2 mgSerum Separators 2Porous Discs 5Column Extenders 2Tris BupH™ Pak (Wash Buffer II) 1

GlutathioneAgarose GST Proteinof Interest

All-in-One™ Mammalian β-Galactosidase Assay KitSingle reagent for cell- or lysate-based assays.

Highlights:• One-reagent formula for cell- or lysate-based assays• Fast and efficient lysis with liquid reagents• No harvesting or washing steps• Flexible protocols for different size cultures

Comparison of Pierce All-in-One™ β-Gal Assay Kit with Brand P β-Gal Assay Kit.

4 Steps 9 Steps

Advantages Offered by the Pierce All-in-One™ β-Gal Assay Kit:1. The Pierce All-in-One™ β-Gal Assay Kit saves time. The average

researcher would carry out a four-step procedure with thePierce system as opposed to a nine-step procedure with theBrand P system.

2. The Pierce All-in-One™ β-Gal Assay Reagent contains the activeingredient M-PER® Mammalian Protein Extraction Reagent, whichallows extraction of 60% more protein than Brand P’s cell lysisreagent. More β-Gal activity can be detected with the PierceAll-in-One™ β-Gal Assay Reagent because cells are lysed efficiently.

Figure 19. Pierce All-in-One™ Assay Reagent surpasses the competition bycombining efficient cell lysis with sensitive ββ-Gal assay. Methods: C6 cellsexpressing β-Galactosidase were grown in 96-well plates and lysed with eitherPierce M-PER® Mammalian Protein Extraction Reagent or Brand P Cell LysisReagent. Either Pierce All-in-One™ Mammalian β-Gal Assay Reagent or Brand P2X Assay Buffer was added to each well, respectively. The reactions werestopped with 150 µl of Stop Solution provided by each kit, and the absorbancewas read at 405 nm using a plate-reading spectrophotometer.

Ordering InformationProduct # Description Pkg. Size75710 All-in-One™ Mammalian β-Galactosidase 3 x 25 ml

Assay Reagent 75707 All-in-One™ Mammalian β-Galactosidase Assay Kit Kit

Includes: All-in-One™ Mammalian β-Galactosidase 25 mlAssay Reagent

M-PER® Mammalian Protein Extraction Reagent 25 mlStop Solution 25 ml

Brand PAll-in-One β-Gal Reagent

2,000

Number of Cells Expressing β-Galactosidase4,000

Abso

rban

ce

10,0008,0006,000

2.0

1.5

1.0

0.5

0

Brand P β-Gal Assay Kit1. Remove media from plate.2. Wash cells twice with PBS.3. Dilute 5X Reporter Lysis Buffer to 1X.4. Add Reporter Lysis Buffer and

incubate for 15 minutes.5. Scrape cells.6. Remove cells to a clean tube, vortex

and centrifuge for 2 minutes at 4˚Cfor 30 minutes.

7. Add 2X Assay Buffer and incubateat 37˚C for 30 minutes.

8. Stop the reactions.9. Measure absorbance at 405 nm.

Pierce All-in-One™ β-Gal Assay Kit1. Remove media from plate.2. Add All-in-One™ β-Gal Assay

Reagent and incubate at 37˚C for 30 minutes.

3. Stop is optional.4. Measure absorbance at 405 nm.

β-Galactosidase Assay Kits

Yeast β-Galactosidase Assay KitIdeal for identifying protein:protein interactions in vivo using two-hybrid systems.

Highlights:• Efficient lysis of yeast cells and a colorimetric detection system • Quantitative or qualitative assay• Allows researcher to test cell cultures directly with no harvesting

and washing steps (ideal for screening applications)• Assay activity from colonies growing on solid media, qualitative

or quantitative, with no re-streaking involved• Can be used with bacterial cells

The gene encoding β-Galactosidase (lacZ) of E. coli has beenwidely used as a reporter gene in many different prokaryotic andeukaryotic organisms. In particular, this gene has proven usefulfor studying gene expression in the yeast S. cerevisiae.

In addition to its utility in studying the regulation of gene expression,the measurement of β-Galactosidase activity can be used to identifyprotein:protein interactions in vivo using two-hybrid systems. Thestrength of the interaction is usually verified and/or quantitatedusing a β-Galactosidase activity assay.

In contrast to methods using 5-bromo-4-chloro-3-indolyl β-D-galac-topyranoside (X-gal) as a β-Galactosidase substrate, our reagentsystem allows for the qualitative or quantitative determination ofβ-Galactosidase activity in solution directly from colonies growing onsolid medium. Part of a colony is picked from a plate and resus-pended in a mixture of Y-PER® Yeast Protein Extraction Reagentand β-Galactosidase assay buffer. After a brief incubation period,the solution turns yellow from the hydrolysis of o -nitrophenyl-β-D-galactopyranoside (ONPG) to o-nitrophenol (ONP) and galactosein a mildly alkaline solution. The assay becomes quantitative if thequantity of cells in the assay is first determined with an absorbancereading taken at 660 nm (OD660).

Figure 20. Linearity of ββ-Galactosidase assay from cells growing in media ina 96-well plate. Strain BRS1002 carrying plasmid pYX122- β-Gal was grown toan OD600 of 1.0, then 100 µl of cells were transferred to a 96-well plate. At time= 0, 100 µl of a 1:1 mixture of lysis reagent and 2X β-Gal assay buffer wereadded to each well and absorbance at 405 nm was determined. Specific activityfor this sample is 160 units (unit=OD405 x 1,000/time/OD660).

Figure 21. Reproducibility of ββ-Galactosidase assay using the novel lysisreagent. The average of three serial dilutions from two independent experimentsperformed weeks apart demonstrates the reproducibility of the assay. In bothexperiments, cells were grown to an OD660 of 0.7, then 100 µl of cells were transferredto a 96-well plate and serially diluted into fresh media. β-Galactosidase activitywas determined after 20 minutes (unit=OD405 x 1,000/time/OD660). In data notshown, Pierce researchers see good reproducibility and the similar specific activ-ity even down to 60,000 cells/well with this expression level. Weaker expressionmay require more cells.

Ordering InformationProduct # Description Pkg. Size75768 Yeast β-Galactosidase Assay Kit Kit

Includes: Y-PER® Yeast Protein Extraction Reagent 25 ml2X β-Galactosidase Assay Buffer 25 ml1M Na2CO3 Stop Solution 25 ml

Experiment 1Experiment 2

Cells/well1 x 106

β-Ga

lact

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ase

Activ

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5 x 105 2.5 x 105

200

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100

50

0

Experiment 1Experiment 2

Average of 3 dilutions of cells

β-Ga

lact

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200

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100

50

0

5

Time (minutes)10

Abso

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20 n

m)

3020 2515

0.45

0.40

0.35

0.30

0.25

0.20

0.15

0.10

0.05

0

y = 0.0151x + 0.0319 R2 = 0.9819


Cell Fra

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β-G

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Cell Fractionation with DetergentsCell fractionation and protein enrichment are important methodsin the rapidly growing field of proteomics. Isolation of subcellularfractions and concentration of proteins in low abundance allowfor more efficient identification and study of proteins of interest,including the isolation of integral membrane proteins andnuclear proteins.

Membrane ProteinsMembrane proteins constituteapproximately 30% of the eukary-otic proteome1 and are a keytarget in drug discovery research.However, they are difficult to isolate

because of their hydrophobicity,basic nature and large size.

Triton® X-114 is a unique detergent thatis used to not only solubilize membrane

proteins, but to also separate them from hydrophilicproteins via phase partitioning at a physiological temperature.2Solutions of Triton® X-114 are homogeneous at 0°C (form a clearmicellar solution) but separate into aqueous and detergent phasesabove 20°C (the cloud point) as micellar aggregates form and thesolution turns turbid. With increased temperature, phase separationproceeds until two clear phases are formed where proteins partitionaccording to their hydrophilic and hydrophobic features. Membraneproteins are enriched in the hydrophobic fraction. Pierce usesthis partitioning method in the Mem-PER® Eukaryotic MembraneProtein Extraction Kit (Product # 89826).3 The protocol involves thegentle lysis of mammalian cells using a mild, proprietary detergentfollowed by membrane protein extraction using the nonionic detergentTriton® X-114. Yeast cells can be fractionated as well using theMem-PER® Kit in combination with glass beads.

Compartmentalization of Proteins in Cell Extract Fractions

Figure 22. The NE-PER® Reagent’s stepwise extraction process results in minimalcross-contamination between cytoplasmic and nuclear fractions. Oct-1 andHsp90 determined by Western blot analysis. β-Gal determined by activity assay.Typical cross-contamination is ≤ 10%.

Nuclear ProteinsDesigned for the stepwise isola-tion of nuclear and cytoplasmicproteins using a mild detergent,the NE-PER® Nuclear andCytoplasmic Extraction Reagents

(Product # 78833) prepare extractsfrom cultured mammalian cells and

tissues by first disrupting the outer cellmembrane to obtain the cytoplasmic

contents and then extracting proteins fromthe nuclei. Cross-contamination between the two fractions is minimal(≤ 10%; Figure 22). With this stepwise fractionation procedure,concentrated nuclear extracts are obtained and gene regulationexperiments are not compromised, as is commonly seen when wholecell lysates are analyzed. Prepared extracts are compatible with manydownstream applications, including electrophoretic mobility shift assays(EMSA) with nuclear extracts, reporter assays with cytosolic extracts,Western blots, enzyme assays, and BCA™ Protein Assays.

Mitochondria Isolation and Protein ExtractionIsolation of mitochondria istypically a laborious processrequiring single-sample processingwith Dounce homogenization.The Mitochondria Isolation Kit

for Cultured Mammalian Cells(Product # 89874) uses a non-

mechanical, reagent-based method formitochondria isolation that allows multiple

samples (six) to be processed concurrently. Cultured mammaliancell pellets are gently lysed using a proprietary formulation (patentpending) that results in maximum yield of mitochondria with minimaldamage to integrity. The product instructions describe guidelines foroptimizing purity vs. yield parameters. The kit also offers an optimizedDounce homogenization procedure, which results in recovery oftwice as much mitochondria (measured as mitochondrial protein)than the reagent-based method. Both methods use differentialcentrifugation to separate the mitochondrial and cytosolic fractionswith a bench-top microcentrifuge and are completed in approximately40 minutes (post-cell harvest). Once isolated, the mitochondriacan be used in downstream applications such as apoptosis,signal transduction and metabolic studies, as well as to facilitatemitochondrial proteomics efforts.

References1. Wallin, E. and Von Heijne, G. (1998). Protein Sci. 7, 1029-1038.2. Bordier, C. (1981). J Biol. Chem. 256, 1604-1607.3. Qoronfleh, W., et al. (2003). Selective enrichment of membrane proteins by partition-

phase separation for proteomic studies. J. Biom. Biotech. 2003(4), 249.

CytoplasmNucleus

Western Blot

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Western BlotActivity Assay

100

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80

70

60

50

40

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0Oct -1 Nuclear

Localized Proteinβ-Gal Cytoplasmic Localized Protein

Hsp90 Cytoplasmic Localized Protein

Cell Fractionation

Mem-PER ® Eukaryotic Membrane Pr

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Nuclear and Cytoplasmic Extrac

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Reag

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Kit

Mitochondria Isolation Kit for Culture

dM

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The Mem-PER® Kit is compatible with many downstream applications.The isolated membrane protein fraction can be used directly inSDS-PAGE and Western blotting (Figure 25). BCA™ Assays, subsequentpurification, etc. can be performed following removal of Reagent Cthrough dialysis using convenient Pierce Slide-A-Lyzer® MINIDialysis Units or Slide-A-Lyzer® Dialysis Cassettes. To effectivelyremove Reagent C and simultaneously maintain protein solubility,perform dialysis overnight at 4°C with a buffer that includes 0.5%detergent. Quantification of extracted membrane proteins with thePierce Micro BCA™ Protein Assay Reagent Kit typically results inapproximately 250 µg of total protein from 5 x 106 C6 cells. Thetotal amount of protein obtained will vary with cell line.

Marker M H M H M H Location

Figure 26. Use of the Mem-PER® Kit to solubilize and isolate yeast integralmembrane proteins (Saccharomyces cerevisiae strain EGY-194). Approximately10-15 mg of wet cell paste was vortexed for 10 minutes at room temperaturein 80 µl of Mem-PER® Reagent A and 100-150 mg of 405-600 micron sizeacid-washed glass beads to disrupt the yeast cell wall. A pulse spin was per-formed to gather the beads, and the cell lysate was transferred to a fresh tubeand kept on ice. The beads were washed with 720 µl of Mem-PER® Reagents Band C, and the wash was then combined with the cell lysate and incubated on icefor 30 minutes. See Figure 23B for the remaining steps. Samples were resolvedon 4-12% Bis-Tris Gels.

References1. Morre, J. and Morre, D. (1989). Preparation of mammalian plasma membranes by

aqueous two-phase partitioning. Biotechniques 7(9), 946-958.2. Lenstra, J.A. and Bloemendal, H. (1983). Topography of the total protein population

from cultured cells upon fractionation by chemical extractions. Eur. J. Biochem. 135,413-423.

3. Ohlendieck, K. (1996). Protein Purification Protocols – Methods in Molecular Biology.Humana Press Inc.: Totowa, NJ. 59, 293-304.

4. Ignacio, R. and Benton, B. (2004). Membrane protein extraction for tissue applications.Mem-PER® Reagents and 2-D Sample Prep for membrane proteins expand to includehard and soft mammalian tissues. Previews, 8(1), 10-11. View it online atwww.piercenet.com or request Literature # 1601010.

5. Stefano, G. B., et al. (2003). Estrogen signaling at the cell surface coupled to nitricoxide release in mytilus edulis nervous system. Endocrinology 144(4), 1234-1240.

Ordering InformationProduct # Description Pkg. Size89826 Mem-PER® Eukaryotic Membrane Protein Kit

Extraction KitIncludes: Mem-PER® Cell Lysis Reagent 10 ml

Mem-PER® Buffer 25 mlMem-PER® Membrane Protein 40 mlSolubilization Reagent

89864 2-D Sample Prep for Membrane Proteins Kit34077 SuperSignal® West Pico 100 ml

Chemiluminescent Substrate23235 Micro BCA™ Protein Assay Reagent Kit Kit

(480 assays)69550 Slide-A-Lyzer® MINI Dialysis Units, 50 units/pkg.

3.5K MWCO, 10-100 µl Sample Capacity89888 PAGEprep® Advance Kit Kit78410 Halt™ Protease Inhibitor Cocktail Kit Kit78415 Halt™ Protease Inhibitor Cocktail Kit, EDTA-Free 1 ml

Visit www.piercenet.com for 2-D Sample Preparation Applications.Pierce has incorporated Mem-PER® Reagents into a new kit(Product # 89864, 2-D Sample Prep for Membrane Proteins)that isolates, concentrates and cleans up membrane proteins.

Integral Membrane Protein of E.R.

Integral Membrane Protein of Mitochondrial

Cytosolic Protein

Peripheral Membrane Protein of Yeast Vacuole

Integral Membrane of Yeast Vacuole

Dpm1p

MP

PGK

VATPase

AP

Mem-PER® Eukaryotic Membrane Protein Extraction KitEfficient, gentle reagents that solubilize and isolate membrane protein in one hour! Mem-PER® Reagents and 2-D Sample Prep for Membrane Proteins expand to include hard and soft mammalian tissues.

Highlights:• Includes an easy and complete protocol that allows for the

isolation of membrane proteins in approximately one hour • Introduces minimal cross-contamination (typically < 10%) of

hydrophilic proteins into the hydrophobic (membrane protein) fraction• Works with a variety of hard and soft tissues (Figures 23A and 24)5

• Works with a variety of eukaryotic cell types [e.g., mammalian(Figure 25) and yeast systems (Figure 26)]

• Provides compatibility with downstream applications includingSDS-PAGE, Western blotting, BCA™ Assays, etc. [use of PierceSlide-A-Lyzer® Dialysis Products and the PAGEprep® Advance Kit(Product # 89888) can aid these procedures]

Isolation of membrane proteins can be a tedious, time-consumingprocess requiring gradient methods and expensive ultracentrifugeequipment1,2 that can be cumbersome and produce poor proteinyields. Ideally, the isolation process should be mild, yet rapid.Recognizing that detergents provide a more convenient method forextraction,3 Pierce researchers developed the Mem-PER® EukaryoticMembrane Protein Extraction Kit, a faster, easier and less expensiveway to isolate membrane proteins. The Mem-PER® Kit consists ofthree reagents developed for the enrichment of integral membraneproteins obtained from cultured eukaryotic cells and tissues.

The simple protocol is accomplished in two parts (Figure 23B). First,cells are lysed with a proprietary detergent and then a secondproprietary detergent is added to solubilize the membrane proteins.Second, the hydrophobic proteins are separated from the hydrophilicproteins through phase-partitioning.4 Following careful separationof the two layers, membrane proteins are ready for subsequentanalysis. Extraction efficiencies of approximately 50% or greaterhave typically been obtained with proteins containing one or twotransmembrane spanning domains. Lower yields may be obtainedwith more complex integral membrane proteins.

Figure 24. Western blots of membrane protein extract prepared from rat liver(panel A) and rat heart (panel B). Hydrophobic (membrane protein) fractionsand corresponding hydrophilic fractions prepared with Mem-PER® Reagents wereanalyzed by Western blot for VDAC (31 kDa, Affinity BioReagents), Cellugyrin(29 kDa, BD Transduction Laboratories) and Flotillin-1 (48 kDa, BD TransductionLaboratories). M: membrane protein fraction and H: hydrophilic fraction.

NIH3T3 HeLa C6Protein M H M H M H Location

Flotillin Membrane 2-Spanner

Cox4 Membrane 1-Spanner

AchE Peripheral

hsp90 Cytosolic

Figure 25. Partitioning of solubilized proteins using Mem-PER® Reagent.Proteins from three cell lines were solubilized and extracted using the Mem-PER®

Kit. Each set of hydrophilic and hydrophobic (membrane protein) fractionsobtained were normalized to one another and analyzed by Western blot for fourproteins from the cellular locations noted. The PAGEprep® Protein Clean-up andEnrichment Kit was used to remove the detergent from the membrane fractionbefore SDS-PAGE/Western analysis of Cox4. A negligible amount of protein wasfound in all debris fractions. Abbreviations: AchE = acetylcholinesterase, Cox4 =cytochrome oxidase subunit 4, hsp90 = heat shock protein 90, M = solubilizedmembrane protein fraction and H = hydrophilic protein fraction.

A

VDAC

M H

Flotillin

BM H

Cellugyrin

Begin with 15 mg of cell paste.

Phase- partitioning

10 minutes at 37°C

TOP = Hydrophilic fraction (save for possible secondary extraction)

BOTTOM = Hydrophobic fraction (membrane proteins – SAVE!)

Solubilized proteins

Insoluble debris is pelleted

3 minutes at 10,000 x g

Spin

2 minutes at 10,000 x g

Spin

Cell suspension: Add 720 ul Reagent B/C. Incubate 30 minutes on ice.

Add 80 ul Reagent A and ~150 mg glass beads. Vortex.

Yeast

Mammalian

Begin with 5 x 106 pelleted cells.

Add 450 ul Reagent B/C.Incubate 30 minutes on ice.

Add 150 ul Reagent A. Incubate 10 minutes at RT.

Figure 23B. Mem-PER® Kit Protocol. Each step of the procedure is outlined for a single extraction of eukaryotic membrane proteins.


Cell Fra

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Cel

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1. Start with 20 mg soft tissue (in 1.5 ml tube) or 20 mg hard tissue ( in 2 ml tube).

3. Soft tissue: Transfer to 2 ml tissue grinder, add 200 µl TBS, grind with 6-10 strokes to homogeneity.

4. Hard tissue: Add 500 µl TBS, cut into small pieces with a razor blade. Homogenize with a hand-held Polytron® Mixer to homogeneity.

6. Discard supernatant and resuspend pellet by pipetting up and down in 150 µl Mem-PER® Reagent A. Follow Mem-PER® Kit protocol as written.

2. Wash with TBS (200 µl soft or 500 µl hard) containing protease inhibitors.

5. Samples from 3 and 4: Transfer each homogenized sample to a clean 1.5 ml microcentrifuge tube, centrifuge at 1,000 x g for 5 minutes at 4˚C.

Figure 23A. Protocol for tissue preparation before membrane protein extraction with Mem-PER® Reagents.

Figure 29. Analysis of cytosolic contamination in mitochondria fraction.Mitochondria and cytosol fractions were prepared from NIH3T3 cells. Eachfraction was analyzed by Western blot for the cytosolic protein, hsp90. M:mitochondria, C: cytosol.

A slight modification of the protocol reduced contamination fromother cellular organelles, although fewer mitochondria were collected.A crude fraction of mitochondria, containing lysosomes and perox-isomes, is prepared with the Mitochondria Isolation Kit. A morepurified preparation of mitochondria was obtained by decreasingthe centrifugation speed used to collect the organelle. Western blotanalysis of PMP70, an abundant integral membrane protein of theperoxisome, and Cathepsin S, a cysteine protease in the lysosome,resulted in > 50% reduction in contamination from these organelles(Figure 30) while recovering approximately 60% of the mitochondriaroutinely collected with a higher centrifugation speed (Table 6).

Figure 30. Reduction of lysosomal and peroxisomal contaminants in mito-chondrial fraction. Mitochondria and cytosol fractions were prepared using amodified reagent-based isolation method. Heavy, more purified mitochondriawere collected at 3,000 x g and the supernatant was centrifuged at 12,000 x gto collect remaining mitochondria. Each fraction was analyzed by Western blotfor A. Peroxisomal membrane protein 70 (PMP70, C6 cells) and B. LysosomalCathepsin S (NIH3T3 cells). M1: 3,000 x g mitochondria fraction, M2: 12,000 x gmitochondria fraction, C: cytosol **See Table 6 for protein quantification.

Table 6. Collection of mitochondria (reagent-based method).

RCF (x g) Protein (µg) % Total Protein 3,000 159.1 62

12,000 98.2 38

Mitochondria collection, normally performed at 12,000 x g, wassplit into a low-speed collection at 3,000 x g and a subsequenthigh-speed collection of remaining mitochondria in the supernatantat 12,000 x g.

The isolated mitochondria may be used in many downstreamapplications, including Western blotting. 2-D Western analysis ofisolated mitochondria was used to identify Mn-Superoxide Dismutase,a detoxifying enzyme residing in the mitochondrial matrix (Figure 31).

Figure 31. 2-D Western blot of superoxide dismutase (Mn-SOD) in isolatedmitochondria. Mitochondria were isolated from NIH3T3 cells using the Douncemethod, resolved by 2-DE and analyzed by Western blot for manganese-containingSOD. Approximately 15 µg of mitochondrial protein was focused on an 11 cm,pH 3-10 IPG strip. The second dimension was performed using 8-16% SDS-PAGE.

Ordering InformationProduct # Description Pkg. Size89874 Mitochondria Isolation Kit for Cultured Cells Kit

Sufficient reagents for 50 applications.Includes: Mitochondria Isolation Kit Reagent A 50 ml

Mitochondria Isolation Kit Reagent B 500 µlMitochondria Isolation Kit Reagent C 70 ml

New! Mitochondria Isolation Kit for TissueThe Mitochondria Isolation Kit for Tissue offers two methods forthe isolation of intact mitochondria from both soft and hard tissues.The first method uses a unique reagent-based procedure thatenables simultaneous multi-sample processing. The second methodrelies on traditional Dounce homogenization for tissue disruptionand subsequent isolation of the organelle. Both procedures relyon differential centrifugation to separate the intact mitochondriausing a bench-top microcentrifuge and are completed in less than60 minutes. Both procedures have been optimized for maximumyield of mitochondria with minimal damage to the integrity of theorganelle. The isolated mitochondria may be used for a number ofdownstream applications, including 2D-MS for proteomics research,disease profiling and metabolic studies.

References for Mitochondrial Proteome1. Lescuyer, P., et al. (2003). Progress in the definition of a reference human mitochondrial

proteome. Proteomics 3, 157-167.1. Taylor, S.W., et al. (2003). Characterization of the human heart mitochondrial proteome.

Nat. Biotechnol. 21, 281-285.

Ordering InformationProduct # Description Pkg. Size89801 Mitochondria Isolation Kit for Tissue Kit

Sufficient reagents for 50 isolations of intact mitochondria from soft and hard tissue.Includes: Mitochondria Isolation Reagent A 50 ml

Mitochondria Isolation Reagent B 500 µlMitochondria Isolation Reagent C 65 mlBSA 235 mgBupH™ Phosphate Buffered Saline 1 pack

A. MW (K)

222 — 114 —88 —50 —

33 —

27 —

17 —

3 10pl

PeroxisomePMP70

Protein Location M1 M2 C

M1 M2 C

LysosomeCathepsin S

A.

B.

Cytosolhsp90

Protein Location M CHighlights:• Fast – isolate intact mitochondria in approximately 40 minutes• Multi-sample format – reagent-based method allows for con-

current preparation of multiple samples• Optional alternate method – reagents and protocol included

for traditional Dounce homogenization• Benchtop-compatibility – isolation performed in a

microcentrifuge tube

The isolation of mitochondria is typically a laborious process requiringsingle-sample processing with Dounce homogenization. The newMitochondria Isolation Kit from Pierce uses a non-mechanical,reagent-based method (Figure 27A) that allows multiple samples(≤ six) to be processed concurrently. Cultured mammalian cellpellets are gently lysed using a proprietary formulation that resultsin maximum yield of mitochondria with minimal damage to integrity.The kit also offers a second isolation method based on traditionalDounce homogenization (Figure 27B), which results in two-foldmore mitochondria recovery, as determined by protein assay. Bothmethods use differential centrifugation to separate the mitochondrialand cytosolic fractions with a bench-top microcentrifuge and arecompleted in approximately 40 minutes (post-cell harvest). Onceisolated, the mitochondria can be used in downstream applicationssuch as apoptosis, signal transduction and metabolic studies, aswell as to facilitate mitochondrial proteomics efforts.

Figure 27. Procedure for the isolation of mitochondria from cultured mammaliancells using (A) the reagent-based method and (B) the Dounce-based method.* A more purified preparation of mitochondria can be obtained by centrifuging at 3,000 x g

instead of 12,000 x g.

Mitochondria isolationThe reagent- and Dounce-basedisolation procedures are outlined inFigure 27A and 27B, respectively.Approximately 2 x 107 mammalian cells(NIH3T3 or C6) were pelleted per sample in a 2 ml microcentrifuge tube. The cells were resuspended inMitochondria Isolation Reagent A and incubated on ice for 2 minutes.Using the reagent-based method, the cells were lysed by addingMitochondria Isolation Reagent B in conjunction with frequentvortexing. The lysate was mixed with Mitochondria IsolationReagent C and centrifuged to remove nuclei, unbroken cells andcellular debris. The supernatant was subsequently centrifuged tocollect the mitochondria, and the pellet was surface-washed toremove cytosolic contaminants. Mitochondria prepared using Douncehomogenization followed a similar protocol. Briefly, 2 x 107 NIH3T3cells were resuspended in Mitochondria Isolation Reagent A,incubated for 2 minutes and then transferred to a tissue grinderand lysed with 80 strokes. The cell homogenate was mixed withMitochondria Isolation Reagent C and the remainder of the protocoldetailed in Figure 27 was performed.

Damage to the outer membrane was assessed by Western blotanalysis of Cytochrome C and voltage-dependent anion channel(VDAC) (Figure 28). Cytochrome C resides in the intermembranespace of undamaged mitochondria and VDAC is an integralmembrane protein in the outer mitochondrial membrane. Negligibleamounts of both proteins were present in the cytosol, indicatingthat the mitochondria remained intact during isolation. Contaminationof the mitochondria with cytosolic components was found to benegligible. Following a single wash of the collected pellet of mito-chondria, minimal heat shock protein 90 (hsp90) contaminationwas detected in a Western blot (Figure 29).

Figure 28. Analysis of mitochondrial integrity. Mitochondria and cytosol fractionswere prepared from C6 cells using the reagent-based method (A and B) or Douncehomogenization (C and D). Fractions were analyzed via Western blot forcytochrome C (A and C) or voltage-dependent anion channel (VDAC) (B and D).SuperSignal® West Pico Chemiluminescent Substrate (Product # 34080) wasused for detection. M: mitochondria, C: cytosol.

Cytochrome C

Reagent-based

A.

C.

B.

D.

M C

Dounce-based

VDAC

M C

Mammalian cells (2 x 107)

Add 800 µl Reagent A

Incubate 2 minutes on ice

Option A. Add 10 µl Reagent B

Option B. Dounce homogenize

Incubate 5 minutes on iceVortex every minute

Add 800 µl Reagent C

Pellet(Nuclei and cell debris) Supernatant

*Centrifuge 12,000 x g 15 minutes at 4°C

Cytosol Mitochondria

Wash with 500 µl Reagent C

Centrifuge 700 x 10 minutes at 4°C

g

Centrifuge 12,000 x g 5 minutes at 4°C


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Mitochondria Isolation Kit forCu

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Mitochondria Isolation Kit for Cultured CellsIsolate intact mitochondria with maximum yield in only 40 minutes.

Blots of HeLa cell cytoplasmic and nuclear fractions were probed forboth Oct-1 (nuclear transcription factor) and Hsp90 (cytoplasmicprotein). Both blots show a minimal amount (generally ≤ 10%) ofcontamination between compartments as determined by densitometricanalysis of the films. Compartmentalization was also evaluated byperforming enzymatic activity assays. Figure 34 shows that 98% ofthe β-Gal activity in the C6 cell line is confined to the cytoplasmicfraction, while 89% of the DNA polymerase activity in HeLa cellsis found in the nuclear fraction. (The 11% DNA polymerase activityfound in the cytoplasmic fraction most likely reflects the presenceof mitochondrial DNA polymerase because mitochondria are foundacross both fractions.) Careful resuspension of the cell pellet withCER I to ensure complete cell lysis in addition to carefully pipettingthe supernatant off the nuclear pellet will maximize the integrityof the extracts.

Figure 34. Compartmentalization of cytoplasmic and nuclear fractions ismaintained using NE-PER® Reagents. A) Extracts from C6 cells (rat glial cellsexpressing β-galactosidase) were assayed for β-Gal activity using a commerciallyavailable kit. B) DNA polymerase activity was assayed using primed M13 single-stranded DNA, buffer, salt and nucleotide composition obtained from theliterature. DNA synthesis was monitored by quantitating the amount of [32P]-dCMP incorporated into TCA-precipitable cpms.

The electrophoretic mobility shift assay (EMSA) is one of thekey applications in the area of gene regulation. Nuclear extractsprepared with NE-PER® Reagents were used with the LightShift®

Chemiluminescent EMSA Kit (Product # 20148).

The volume of nuclear extract used in these reactions did notexceed 2 µl (10% of total reaction volume). If the protein of interestis less abundant, the concentration of the nuclear extract can beincreased by decreasing the volume of NE-PER® Extraction Reagentused during extraction. If it still is necessary to use larger volumes ofextract for an EMSA, Pierce Slide-A-Lyzer® MINI Dialysis Units can beused to remove interfering substances in the NER Nuclear Reagent.

Figure 35. Chemiluminescent EMSA of four different DNA-protein complexes.DNA binding reactions were performed using 20 fmol biotin-labeled DNA duplex(1 biotin per strand) and 2 µl (6.8 µg total protein) NE-PER® Nuclear Extract pre-pared from HeLa cells. For reactions containing specific competitor DNA, a 200-fold molar excess of unlabeled specific duplex was used.

References1. Adilakshmi, T. and Laine, R.O. (2002). Ribosomal protein S25 mRNA partners with MTF-1

and La to provide a p53-mediated mechanism for survival or death. J. Biol. Chem. 277(6),4147-4151.

2. Liu, H.B., et al. (2003). Estrogen receptor alpha mediates estrogen’s immune protectionin autoimmune disease. J. Immunol. 171(12), 6936-6940.

3. Sanceau, J. (2002). Interferons inhibit tumor necrosis factor-α-mediated matrixmetalloproteinase-9 activation via interferon regulatory factor-1 binding competitionfor NF-κβ. J. Biol. Chem. 277, 35766-35775.

4. Current Protocols in Molecular Biology (1993).5. Zerivitz, K. and Akusjarvi, G. (1989). Gene Anal. Tech. 6, 101-109.6. Dignam, J.D., et al. (1983). Nucleic Acids Res. 11, 1475-1489.

Ordering InformationProduct # Description Pkg. Size78833 NE-PER® Nuclear and Cytoplasmic Extraction Kit Kit

Sufficient reagents for extracting 50 cell pellet fractions having packed cell volumes of 20 µl each (a total of ~2.0 g of cell paste).Includes: Cytoplasmic Extraction Reagent I (CER I) 10 ml

Cytoplasmic Extraction Reagent II (CER II) 550 µlNuclear Extraction Reagent (NER) 5 ml

89863 2-D Sample Prep for Nuclear Proteins KitSufficient reagents for 25 applicationsNE-PER® Nuclear and Cytoplasmic Extraction Reagents:Cytoplasmic Extraction Reagent I (CER I) 5 mlCytoplasmic Extraction Reagent II (CER II) 0.275 mlNuclear Extraction Reagent (NER) 2.5 ml

2-D Sample Buffer for Nuclear Proteins:2-D Sample Buffer for Nuclear Proteins, Component A 18 ml2-D Sample Buffer for Nuclear Proteins, Component B 16.5 g

Protein Desalting Spin Columns 25 columns

Extract - + +Competitor - - +

- + +- - +

- + +- - +

- + +- - +

EBNA Oct-1 AP1 NF-κB

CytoplasmicNuclear

1

A. β-Galactosidase

β-gal Sample2

Tota

l Act

ivity

(mU)

53 4

120

100

80

60

40

20

0

Cyto

Nuclear

0

B. DNA Polymerase

Time (minutes)15

CPM

7530 45 60

80,000

70,000

60,000

50,000

40,000

30,000

20,000

10,000

0

NE-PER® Nuclear and Cytoplasmic Extraction KitA fast and easy means of obtaining concentrated nuclear extracts that can be used in a variety of downstream studies.

Highlights:• Fast – obtain nuclear and cytoplasmic fractions in less than two hours• Easy – eliminate freeze/thaw cycles, Dounce homogenization,

lengthy centrifugation times and cold room work• Versatile – obtain nuclear and cytoplasmic extracts separately

from the same set of cells or tissue1,2

• Compatible with downstream assays including Western blotting,gel-shift assays, protein assays, reporter gene assays andenzyme activity assays1-3

The preparation of good nuclear protein extracts is central to thesuccess of many gene regulation studies. Nuclear extracts areused instead of whole cell lysates for the following reasons. First,many experiments in the area of gene regulation are adverselyaffected by cellular components present in whole cell lysates.Second, the concentration of the nuclear protein of interest isdiluted by the vast array of cytoplasmic proteins present in wholecell extracts. Finally, whole cell lysates are complicated by thepresence of genomic DNA and mRNA.

A variety of methods exist to isolate nuclei and prepare nuclearprotein extracts.4-6 Most of these are, however, lengthy processesrequiring mechanical homogenization, freeze/thaw cycles, extensivecentrifugation or dialysis steps that may compromise the integrityof many fragile nuclear proteins. Pierce developed NE-PER® Nuclearand Cytoplasmic Extraction Reagents, which enable a stepwise lysisof cells that generates both functional cytoplasmic and nuclearprotein fractions in less than two hours.

Figure 32 shows the results of Micro BCA™ Protein Assays performedon both the cytosolic and nuclear fractions prepared from a varietyof cell lines.

Figure 32. Total protein profile of cytoplasmic and nuclear extracts preparedfrom a variety of mammalian cell lines using NE-PER® Reagents. Protein wasquantitated using Pierce Micro BCA™ Protein Assay Reagent (Product # 23235).Numbers shown are the average of two separate isolations.

The data show that the yield of protein in the cytoplasmic extractis cell-line dependent, with the larger cell sizes such as HeLa andHepa 1-6 giving more total protein (500 µg) compared to the smallerNIH3T3 fibroblasts and rat C6 brain cells (250 µg). Nuclear proteinyields averaged 100-200 µg total protein from 2 x 106 cells at aconcentration on the order of 1.5 mg/ml, independent of cell type.The protein concentration of the nuclear extracts can be manipulatedeasily by varying the volume of nuclear extraction reagent (NER) usedin the extraction without any significant loss in extraction efficiency.

The key to success for the NE-PER® Kit is the stepwise isolation ofcytoplasmic and nuclear fractions, so it is important to maintain theintegrity of the two cellular compartments. Western blot analyseswere used to assess the level of cross-contamination between thetwo fractions (Figure 33).

Figure 33. Western blot analyses of HeLa cytoplasmic and nuclear fractionsillustrating the low level of cross-contamination. Four samples were analyzedby loading equivalent amounts of total protein (20 µg) in each lane of a 4-20%gradient Tris-Glycine denaturing gel. The protein was transferred to nitrocelluloseand blocked with 3% BSA in Tris buffered saline. A) Blot probed with Oct-1antiserum (1:400) followed by 1:100,000 secondary antibody-horseradishperoxidase (HRP) conjugate. B) Blot probed with Hsp90 antiserum (1:400)followed by 1:50,000 secondary antibody-HRP conjugate. Both blots weredeveloped using Pierce SuperSignal® West Pico Chemiluminescent Substrate(Product # 34080).

A.

Oct 1

C N C N C N C NkD250 148

60

42 30 22 17

B.

Hsp90

C N C N C N C NkD

250 148

60 42 30 22 17Cytoplasmic

Nuclear

HeLa Cos7

µg o

f Pro

tein

s

C63T3 Hepa

600

500

400

300

200

100

0


Cell Fra

ctionation


Cel

l Fra

ctio

natio

n

Y-DER™ Yeast DNA Extraction Kit Extracts and purifies genomic and plasmid DNA from yeast in less than one hour.

Highlights:• Eliminates the need for glass beads or harsh enzyme treatments• Prepares DNA for polymerase chain reaction (PCR) amplification• Rapidly isolates plasmid DNA from Saccharomyces cerevisiae

suitable for transformation of Escherichia coli• Scalable kit – from single colonies to 500 ml culture

Current protocols for the extraction and purification of DNA from yeastare time-consuming and labor-intensive. The yeast cell is notoriouslydifficult to lyse due to a very complex proteinaceous cell wall thatprovides rigidity to the relatively weak plasma membrane. The Y-DER™

Reagent Kit uses Y-PER® Reagent to quickly and easily lyse yeast cells.

The Y-DER™ Reagent Kit surpasses the historical methods of DNAisolation from yeast. The protocol requires less than one hour,works without enzymatic treatment or glass beads, and yields littleto no RNA contamination.

In studies with S. cerevisiae, Y-DER™ Reagent consistently obtainshigh yields of genomic and plasmid DNA. Y-DER™ Reagent-purifiedDNA is suitable for PCR amplification (Figure 36), bacterial trans-formations (both chemical and electrocompetent cells), restrictiondigestions, and hybridization applications.

1 2 3 4 5

Ordering InformationProduct # Description Pkg. Size78870 Y-DER™ Yeast DNA Extraction Kit Kit

Sufficient reagents for 50 purifications from 10 ml cultures.Includes: Y-PER® Yeast Protein Extraction Reagent 25 ml

DNA Releasing Agent A 20 mlDNA Releasing Agent B 20 mlProtein Removal Reagent 10 ml

Figure 36. Examples of Y-DER™ Reagent extraction ofyeast genomic DNA and subsequent PCR amplifications.DNA was extracted from S. cerevisiae strain DY150transformed with a plasmid harboring the gene for greenfluorescent protein (GFP) and purified DNA was used toamplify the chromosomal ACT1 and UME6 genes and thegene-encoding GFP carried on the plasmid. Lane 1. LambdaDNA digested with Hind III, Lane 2. S. cerevisiae genomicDNA (smaller band approx. 5 kb corresponds to dsRNAfrom the yeast killer virus), Lane 3. PCR amplification ofGFP from plasmid, Lane 4. PCR amplification of chromo-somal ACT1 gene, and Lane 5. PCR amplification ofchromosomal UME6 gene.

Lyse-N-Go™ PCR ReagentFacilitates the release of PCR-ready DNA in less than 10 minutes.

Highlights:• Perform the PCR directly from lysed bacteria, yeast (tested on

Saccharomyces cerevisiae), cultured cells, blood, tissue and plants• Eliminates the need for expensive and time-consuming DNA

purification kits• Lyse and amplify in a single tube, reducing the risk of

contamination and handling problems• Simple, efficient and fast protocol is ideal for

high-throughput screening

Polymerase chain reaction (PCR) technology has enabled rapidanalysis of DNA sequences and opened doors to further scientificinquiry. Because DNA amplification can occur in the presence ofother cellular components such as proteins, RNA and nonspecificDNAs, it is possible to amplify directly from bacterial cell lysateswithout first purifying the DNA. An amplification-compatiblereagent is all one needs to lyse the cell and release the templateDNA. Lyse-N-Go™ PCR Reagent releases DNA from cells in a formready for PCR amplification.

Experiments performed demonstrate the ability of Lyse-N-Go™ PCRReagent to provide amplification-ready plasmid DNA for the PCR. Theproprietary formulation has been tested and found to be compatiblewith several commercially available Taq polymerases. It has alsobeen used to lyse other types of cells including yeast colonies, someanimal and plant tissues, whole blood, and cultured mammalian cellsfor direct PCR application. The versatility of the Lyse-N-Go™ PCRReagent provides a very useful tool in molecular biology research.

Positive colonies DNA template Negative coloniesM 1 2 3 M 1 2 3 M 1 2 3

Figure 37. Amplification of Escherichia coli colonies by PCR after cell lysis withLyse-N-Go™ PCR Reagent. E. coli JM-109 cells transformed with pUSE vectorwith or without a 0.8 kb cDNA insert (rat urate oxidase), respectively, were grownovernight at 37°C on LB plates. A proportion of the colonies were picked up bya pipette tip and suspended in 5 µl of Lyse-N-Go™ PCR Reagent. The suspensionis then heated at 95°C for 2 minutes before adding the amplification mixture. ThePCR reaction was carried out for 30 cycles at 94°C for 30 seconds, 55°C for45 seconds and 68°C for 60 seconds. At the same time, 10 ng of pUSE vectorcontaining the same insert were used as a positive control. The amplified productswere analyzed on a 1% agarose gel by electrophoresis. Lane M is the KiloBaseDNA marker. Reactions were run in triplicate.

Ordering InformationProduct # Description Pkg. Size78882 Lyse-N-Go™ PCR Reagent 10 ml

Properties and Types of DetergentsDetergents are amphipathic molecules, meaning they contain botha nonpolar “tail” having aliphatic or aromatic character and a polar“head” (Figure 38). Ionic character of the polar head group formsthe basis for broad classification of detergents; they may be ionic(charged, either anionic or cationic), nonionic (uncharged), orzwitterionic (having both positively and negatively charged groupsbut with a net charge of zero). Like the components of biologicalmembranes, detergents have hydrophobic-associating propertiesas a result of their nonpolar tail groups. Nevertheless, detergentsare themselves water-soluble. Consequently, detergent moleculesallow the dispersion (miscibility) of water-insoluble, hydrophobiccompounds into aqueous media, including the extraction and sol-ubilization of membrane proteins.

Figure 38. Schematic structure of a detergent (top) and the complete structureof CHAPS (bottom), an example of a zwitterionic detergent.

Detergents at low concentration in aqueous solution form amonolayer at the air-liquid interface. At higher concentrations,detergent monomers aggregate into structures called micelles(Figure 39). A micelle is a thermodynamically stable colloidalaggregate of detergent monomers wherein the nonpolar ends aresequestered inward, avoiding exposure to water, and the polarends are oriented outward in contact with the water.

Figure 39. Idealized structure of a detergent micelle.

Both the number of detergent monomers per micelle (aggregationnumber) and the range of detergent concentration above whichmicelles form (called the critical micelle concentration, CMC) areproperties specific to each particular detergent (Table 7, page 29).The critical micelle temperature (CMT) is the lowest temperatureat which micelles can form. The CMT corresponds to what is knownas the cloud point because detergent micelles form crystallinesuspensions at temperatures below the CMT and are clear againat temperatures above the CMT.

Detergent properties are affected by experimental conditions suchas concentration, temperature, buffer pH and ionic strength, andthe presence of various additives. For example, the CMC of certainnonionic detergents decreases with increasing temperature, whilethe CMC of ionic detergents decreases with addition of counter ionas a result of reduced electrostatic repulsion among the chargedhead groups. In other cases, additives such as urea effectivelydisrupt water structure and cause a decrease in detergent CMC.Generally, dramatic increases in aggregation number occur withincreasing ionic strength.

Detergents can be denaturing or non-denaturing with respect toprotein structure. Denaturing detergents can be anionic, such assodium dodecyl sulfate (SDS), or cationic, such as ethyl trimethylammonium bromide. These detergents totally disrupt membranesand denature proteins by breaking protein:protein interaction.Nondenaturing detergents can be divided into nonionic detergentssuch as Triton® X-100, bile salts such as cholate, and zwitterionicdetergents such as CHAPS.

Protein Binding and SolubilizationDenaturing detergents such as SDS bind to both membrane(hydrophobic) and nonmembrane (water-soluble, hydrophilic)proteins at concentrations below the CMC; i.e. monomers. Thereaction is equilibrium-driven until saturated. Therefore, the freeconcentration of monomers determines the detergent concentration.SDS binding is cooperative (the binding of one molecule of SDSincreases the probability that another molecule of SDS will bindto that protein) and alters most proteins into rigid rods whoselength is proportional to molecular weight.

Nondenaturing detergents such as Triton® X-100 have rigid and bulkynonpolar heads that do not penetrate into water-soluble proteins;consequently, they generally do not disrupt native interactions andstructures of water-soluble proteins and do not have cooperativebinding properties. The main effect of nondenaturing detergents isto associate with hydrophobic parts of membrane proteins, therebyconferring miscibility to them.

At concentrations below the CMC, detergent monomers bind towater-soluble proteins. Above the CMC, binding of detergent toproteins competes with the self association of detergent moleculesinto micelles.1 Consequently, there is effectively no increase inprotein-bound detergent monomers with increasing detergentconcentration beyond the CMC.

HO OH

OH O

N+

SO

O

O–

CHAPSM.W. 614.88

NH

Hydrophobic“Tail”

Hydrophilic“Head”


Deterg

ents


DN

AEx

tract

ion

DetergentsDNA Extraction

Detergent monomers solubilize membrane proteins by partitioninginto the membrane bilayer. With increasing amounts of detergents,membranes undergo various stages of solubilization. The initialstage is lysis or rupture of the membrane. At detergent:membranelipid molar ratios of 0.1:1 through 1:1, the lipid bilayer usually remainsintact but selective extraction of some membrane proteins occurs.Increasing the ratio to 2:1, solubilization of the membrane occurs,resulting in mixed micelles. These include phospholipid-detergentmicelles, detergent-protein micelles and lipid-detergent-protein micelles.At a ratio of 10:1, all native membrane lipid:protein interactionsare effectively exchanged for detergent:protein interactions.

The amount of detergent needed for optimal protein extractiondepends on the CMC, aggregation number, temperature and natureof the membrane and the detergent. The solubilization buffer shouldcontain sufficient detergent to provide greater than 1 micelle permembrane protein molecule to ensure that individual proteinmolecules are isolated in separate micelles.2

Removal of Detergent from Solubilized ProteinsHowever necessary and beneficial the use of detergent may have beenfor initial cell lysis or membrane protein extractions, subsequentapplications or experiments with the extracted proteins may requireremoval of some or all of the detergent. For example, althoughmany water-soluble proteins are functional in detergent-solubilizedform, membrane proteins are often modified and inactivated bydetergent solubilization as a result of native lipid interactions havingbeen disrupted. In some such cases, membrane protein functionis restored when they are reconstituted into bilayer membranes byreplacement of detergent with phospholipids or other membrane-like lipid mixtures. The function of an individual protein can bestudied in isolation if it is first purified and then reconstituted intoan artificial membrane (although recovery of native orientation inthe membrane is a major challenge). Even where restoration ofprotein function is not an issue, detergent concentration may haveto be decreased in a sample to make it compatible with proteinassays or gel electrophoresis.

Detergent removal can be attempted in a number ways. Dialysisis effective for removal of detergents that have very high CMCsand/or small aggregation numbers, such the N -octyl glucosides.Detergents with low CMCs and large aggregation numbers cannotbe dialyzed since most of the detergent molecules will be in micelles

that are too large to diffuse through the pores of the dialysismembrane; only excess monomer can be dialyzed. Ion exchangechromatography using appropriate conditions to selectively bind andelute the proteins of interest is another effective way to removedetergent.3 Sucrose density gradient separation also can be used.4

In addition to SnakeSkin® Dialysis Tubing and Slide-A-Lyzer® DialysisCassettes and MINI Dialysis Units, Pierce offers two unique productsdesigned specifically to assist in detergent removal. SDS-Out ™

Sodium Dodecyl Sulfate Precipitation Reagent (Product # 20308and 20310) enables efficient cold-precipitation of excess SDS fromprotein solutions. Extracti-Gel® D Detergent Removal Gel (Product# 20208 and 20346) allows fairly selective affinity-based removal ofmany different detergents from solutions. Pierce also offers Zeba™

Protein Desalt Spin Columns (Product # 89882 and 89889) for theremoval of detergent from small volume samples and the PAGEprep®

Advance Kit (Product # 89888) for removal of detergents and othercontaminants from protein samples prior to analysis by SDS-PAGE.

Surfact-Amps® Purified Detergent SolutionsAlthough detergents are available from several commercial sourcesand used routinely in many research laboratories, the importance ofdetergent purity and stability is not widely appreciated. Detergentsoften contain trace impurities from their manufacture. Some ofthese impurities, especially peroxides that are found in most nonionicdetergents, will destroy protein activity.5,6 In addition, several typesof detergents oxidize readily when exposed to the air or UV light,causing them to lose their properties and potency as solubilizingagents. Pierce offers several high-purity, low peroxide-containingdetergents that are packaged under nitrogen gas in clear glassampules. These Surfact-Amps® Purified Detergent Solutions pro-vide unsurpassed convenience, quality and consistency for alldetergent applications. The Surfact-Pak™ Detergent Sampler Kit(Product # 28340) includes 10 different purified detergents (sevenin the Surfact-Amps® Format and three in solid form).

References1. Helenius, A. and Simons, K. (1975). Biochim. Biophys. Acta 415, 29-79.2. Helenius, A., et al. (1979). Methods Enzymol. 56, 734-749.3. Hjelmeland, L.M. (1990). Methods Enzymol. 182, 277-282.4. Warren, G.B., et al. (1974). Proc. Nat. Acad. Sci. USA 71, 622-626.5. Chang, H.W. and Bock, E. (1980). Anal. Biochem. 104, 112-117.6. Ashani, Y. and Catravas, G. (1980). Anal. Biochem. 109, 55-62.

Table 7. Properties of common detergents.

Detergent Description Aggregation Number Micelle MW MW CMC (mM) CMC % w/v Cloud Point (°C) DialyzableTriton® X-100 Nonionic 140 90,000 647 0.24 0.0155 64 NoTriton® X-114 Nonionic — — 537 0.21 0.0113 23 NoNP-40 Nonionic 149 90,000 617 0.29 0.0179 80 NoBrij®-35 Nonionic 40 49,000 1225 0.09 0.1103 > 100 NoBrij®-58 Nonionic 70 82,000 1120 0.077 0.0086 > 100 NoTween®-20 Nonionic — — 1228 0.06 0.0074 95 NoTween®-80 Nonionic 60 76,000 1310 0.012 0.0016 — NoOctyl Glucoside Nonionic 27 8,000 292 23-25 0.6716-0.7300 > 100 YesOctylthio Glucoside Nonionic — — 308 9 0.2772 > 100 YesSDS Anionic 62 18,000 288 6-8 0.1728-2304 > 100 YesCHAPS Zwitterionic 10 6,149 615 8-10 0.4920-0.6150 > 100 YesCHAPSO Zwitterionic 11 6,940 631 8-10 0.5048 90 Yes

Surfact-Amps® Purified Detergent SolutionsNonionic detergents – ready-to-use solutions. Nothing could be easier … nothing protects better!

Highlights:• Precise 10% detergent solutions in ultrapure water• Easy to accurately dispense and dilute for use• Exceptionally pure – less than 1.0 µeq/ml peroxides and carbonyls• Maximal stability and long shelf life – packed in glass ampules

under inert nitrogen gas

Don’t begin your experiment with detergents of unknown ageand purity! Surfact-Amps® Purified Detergent Solutions provideunsurpassed purity, quality and stability in convenient 10% solutions.Unlike neat detergent formulations, Surfact-Amps® 10% Solutionsare not so viscous that you cannot aliquot them accurately. Justopen an ampule and dilute the contents into your buffer at thedesired concentration.

Surfact-Pak™ Detergent Sampler and Surfact-Amps® DetergentsConvenient 10-sample package of detergents allows for trial testing and experimentation.

Ordering InformationProduct # Description Pkg. Size28340 Surfact-Pak™ Detergent Sampler Kit

Contains: Surfact-Amps® Purified DetergentsSurfact-Amps® X-100 10 mlSurfact-Amps® 35 10 mlSurfact-Amps® 20 10 mlSurfact-Amps® NP-40 10 mlSurfact-Amps® 80 10 mlSurfact-Amps® X-114 10 mlSurfact-Amps® 58 10 ml

Octyl β-Glucoside 100 mgOctyl β-Thioglucopyranoside 100 mgCHAPS 100 mg

Tween®-based Detergents

28320 Surfact-Amps® 20 6 x 10 ml(Active Ingredient: Tween®-20)

28328 Surfact-Amps® 80 6 x 10 ml(Active Ingredient: Tween®-80)

Product # Description Pkg. Size

Triton®-based Detergents

28314 Surfact-Amps® X-100 6 x 10 ml(Active Ingredient: Triton® X-100)

28332 Surfact-Amps® X-114 6 x 10 ml(Active Ingredient: Triton® X-114)

Nonidet-based Detergent

28324 Surfact-Amps® NP-40 6 x 10 ml(Active Ingredient: Nonidet P-40)

Brij®-based Detergents

28316 Surfact-Amps® 35 6 x 10 ml(Active Ingredient: Brij®-35)

28336 Surfact-Amps® 58 6 x 10 ml(Active Ingredient: Brij®-58)

20150 Brij®-35, 30% Solution 950 mlActive ingredient is supplied as a purified 10% aqueous solution ampuled under nitrogen.


Deterg

ents


Det

erg

ents

CHAPS & CHAPSOZwitterionic detergents ideal for protecting the native state of proteins.

Highlights:• Able to disrupt nonspecific protein interactions• Less protein aggregation than nonionic detergents• Electrically neutral and nondenaturing• Easily removed by dialysis

Ordering InformationProduct # Description Pkg. Size28300 CHAPS 5 g

(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)

28299 CHAPS 100 g28304 CHAPSO 5 g

(3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate)

CHAPSOM.W. 630.88

HO OH

OH O

N+

SO

O

O–

NH

HO

CHAPSM.W. 614.88

Octyl β-Glucoside A nonionic detergent widely used for membrane protein solubilization.

Highlights:• Low molecular weight permits easy removal by dialysis

Ordering InformationProduct # Description Pkg. Size28309 Octyl β-Glucoside 1 g28310 Octyl β-Glucoside 5 g

OH

HO

HHO

H

HOHH

O

HO

Octyl β-GlucosideM.W. 292.37

Octyl β-ThioglucopyranosideOffers specific stability over Octyl β-Glucoside.

Highlights:• Not affected by β-glucosidase, which is an enzyme found in

some biological systems• Optically transparent and dialyzable• Offers solubilizing power comparable to Octyl β-Glucoside, with

increased stability

Ordering InformationProduct # Description Pkg. Size28351 Octyl β-Thioglucopyranoside 5 g

(OTG)

Sodium Dodecyl Sulfate (SDS) C12

Great for applications in which renaturation is important.

Highlights:• Greater than 98% C12 alkyl sulfate, with low levels of hexadecyl

sulfate C16, which can inhibit protein renaturation

Ordering InformationProduct # Description Pkg. Size28312 SDS, C12 500 g

(Sodium dodecyl sulfate, C12)

Sodium Dodecyl Sulfate (SDS)The ideal detergent when resolution is most important.

Highlights:• Unique distribution of carbon chain lengths in Pierce SDS (lauryl) is

advantageous in resolving viral proteins during gel electrophoresis• Can be used when renaturation after SDS-PAGE is required (if gels

are treated according to the procedure of Blank, et al. to removeC14 and C16 alkyl sulfates)1

Reference1. Blank, A., et al. (1980). Federation Proceedings 39(6), Abstracts ABSC/TBS. Abstract

no. 1285, 1951.

Ordering InformationProduct # Description Pkg. Size28364 SDS 100 g

(Sodium dodecyl sulfate, lauryl)Typical Analysis – C12: 63.5%, C14: 29.5%, C16: 7.0%

28365 SDS 1 kg

Extracti-Gel® D Detergent Removing GelMakes detergent removal efficient, fast and easy with high protein recoveries, too.

Highlights:• Allows relatively small detergent molecules to enter the gel

matrix where they interact with a specially developed ligandcapable of removing them from solution

• Low exclusion limit of the support decreases the possibility ofnonspecific binding

• Recommended for use with biological macromolecules that aregreater than 10 kDa

• Detergent is extracted without losing valuable protein• Reusable affinity matrix can be regenerated up to three times• Compatible with a wide variety of buffers (pH 3.5-10)• Recovery of dilute protein solutions enhanced by use of a

carrier protein

Table 8. Detergent binding data.

CapacityDetergent Product # (mg/ml gel) Binding ConditionsBrij®-35 28316 80 100 mM Phosphate Buffer, pH 7.0CHAPS 28300 50 0.05 M Tris Buffer, pH 9.0SDS 28312 80 0.05 M Tris Buffer, pH 9.0Triton® X-100 28314 57 100 mM Phosphate Buffer, pH 7.0Tween®-20 28320 74 100 mM Phosphate Buffer, pH 7.0

Ordering InformationProduct # Description Pkg. Size20208 Extracti-Gel® D Detergent Removing Gel 10 ml20303 Extracti-Gel® D Detergent Removing Gel 100 ml20346 Extracti-Gel® D Detergent Removing Gel 5 x 1 ml

(Pre-packed Columns)

SDS-Out™ Sodium Dodecyl Sulfate Precipitation ReagentThe solution to the small-sample SDS removal problem.

Highlights:• Convenient removal of SDS from microliter-volume samples• Use for samples containing 0.1-1% SDS• Minimal dilution of small-volume samples• Protein sample recovery in 90%-100% range tested for proteins

such as BSA, cytochrome C, soybean trypsin inhibitor, ovalbumin,ribonuclease A, myoglobin and human IgG

• Kit contains spin cup and collection tube accessories for usewith the SDS-Out™ SDS Precipitation Reagent

Table 9. Protein recovery after removal of SDS from protein solution with SDS-Out™ SDS Precipitation Reagent.

Initial Concentration of SDSProtein* 0.125% 0.25% 0.5% 1.0%

% Protein % Protein % Protein % ProteinRecovery Recovery Recovery Recovery

Bovine Serum Albumin 97.5 98.0 98.4 100Cytochrome C 100 100 100 100Soybean Trypsin Inhibitor 99.0 98.5 98.8 100Ovalbumin 99.8 99.6 98.1 93.8Ribonuclease A 100 100 100 100Myoglobin 100 100 100 97.9Human IgG 100 92.7 88.7 76.2* 0.5 ml samples of protein solution each at a concentration of 1 mg/ml were used to

develop this table.

Ordering InformationProduct # Description Pkg. Size20308 SDS-Out™ SDS Precipitation Kit Kit

Sufficient reagent to precipitate SDS from 200 ml of protein solution.Includes: SDS Precipitation Reagent 10 ml

Micro Sample Tubes (graduated) 12 x 1.5 mlHandee™ Spin Cup Columns 12 ea.0.45 µm cellulose acetate

20310 SDS-Out™ SDS Precipitation Reagent 25 ml


Det

erg

ents


Deterg

ents

All living organisms contain proteolytic enzymes (proteases andpeptidases). Proteases are required for a variety of cellular functions,such as cellular repair or the digestion of extracellular material. Inwhole cells, protease activity is tightly regulated by compartmen-talization or inhibitors to prevent damage to cellular proteins. Celllysis disturbs this regulation and proteolytic degradation of thesample becomes a concern. Therefore, addition of protease inhibitorsto cell lysis buffers is often required.

Protease inhibitors are biological or chemical compounds thatfunction by reversibly or irreversibly binding to the protease.Proteases generally belong to one of four evolutionarily distinctenzyme families based on the functional groups involved in cleavageof the peptide bond (Table 10). Therefore, several different types ofinhibitors are generally required to protect proteins from proteolysisduring extraction and purification.

Although the particular suite of protease inhibitors necessary toprevent degradation of proteins of interest may vary, Halt™ ProteaseInhibitor Cocktails (Product # 78410 and 78415) exhibit excellentinhibition of a variety of protease activities. The cocktails are suitable

for the protection of proteins during extraction from animal tissues,plant tissues, yeast or bacteria and are compatible with Poppers™

Protein Extraction Reagents. Pierce also offers the protease inhibitorPMSF (Product # 36978).

Table 10. Families of proteases and their inhibitors.

Family Example Enzymes Inhibitors*Serine protease Trypsin Phenylmethlysulfonyl fluoride (PMSF)

Subtilisin ChloromethylketonesThrombin BenzamidineProtease factor Xa Aprotinin

Thiol protease Papain Iodoacetate(Cysteine protease) Calpain I and II Leupeptin

CathepsinsCaspase-1

Acid proteases Pepsin Pepstatin A(Carboxyl or Rennin aspartyl proteases) Cathepsin DMetalloproteases Carboxylpeptidase A EDTA(Zinc proteases) Collagenase 1,10 Phenanthroline

Thermolysin* Inhibitors do not affect all members of a protease family

Halt™ Protease Inhibitor CocktailsCompatible protease inhibitors for all occasions!

1 2 3

Halt™ Protease Inhibitor Cocktails inhibit a variety of proteases. Bothready-to-use formulations inhibit serine, cysteine and calpain proteasesand metalloproteases. The EDTA-free formulation is ideal for preparingsamples that will be analyzed by 2-D gel electrophoresis.

Halt™ Cocktails are easy to use. Add the appropriate volume ofinhibitor cocktail to the sample (10 µl per 1 ml of cell extract)and continue with your purification. The Halt™ Protease InhibitorCocktails are compatible with Poppers™ Reagents and can beused with animal cells and tissues, bacteria, plants and yeast.

Ordering InformationProduct # Description Pkg. Size78410 Halt™ Protease Inhibitor Cocktail Kit Kit

Sufficient reagents for 200 ml of extract.78415 Halt™ Protease Inhibitor Cocktail, EDTA-Free 1 ml

Sufficient reagent for 100 ml of extract.

Figure 40. SDS-PAGE analysis of the inhibition ofprotease activity using Halt™ Protease InhibitorCocktails. Bovine serum albumin (BSA) was incubatedovernight at 37°C in the presence of the proteasepapain. Lane 1. digestion of BSA in the absence ofprotease inhibitors. Lane 2. BSA digestion in thepresence of Halt™ Protease Inhibitor Cocktail. Lane 3.BSA digestion in the presence of Halt™ Protease InhibitorCocktail, EDTA-Free.

PMSFReacts with serine residues to inhibit trypsin, chymotrypsin, thrombin and papain.

Ordering InformationProduct # Description Pkg. Size36978 PMSF 5 g

(Phenylmethylsulfonyl fluoride)

S

F

O

OPMSF

M.W. 174.19

Soybean Trypsin Inhibitor, ImmobilizedFor effective removal of trypsin, chymotrypsin and elastase from protein digests.

Ordering InformationProduct # Description Pkg. Size20235 Immobilized Soybean Trypsin Inhibitor 2 ml gel

STI coupled to spherical 4% beaded agarose.Capacity: Minimum of 6 mg trypsin per ml of gelSupplied in glycerol containing 0.05% NaN3

Pro-Matrix™ Protein Refolding KitBring your protein back into the fold!

Highlights:• Robust – conditions and components examined are limited to

those having the most significant and general utility as fold-ing buffers

• Convenient – three-level matrix design significantly reducesthe amount of secondary optimization required and increasesthe ease of data interpretation

• Adjustable matrix format – allows refolding experiments to becustomized to the target protein; known positive and negativeinteractions between buffer components are addressed, minimizingunnecessary analyses

• High-purity reagents – reagents are formulated using stringentstandards so that consistent results are attained

Inclusion bodies, which consist of misfolded insoluble proteinaggregates, are often the products of overexpressing recombinantproteins in bacterial hosts. Although many proteins can be refolded,it is typically a difficult and time-consuming process to create theproper conditions to restore the native conformation. The Pro-Matrix™

Protein Refolding Kit is designed to simplify the development of ahigh-yield/high-concentration refolding protocol.

The Pro-Matrix™ Protein Refolding Kit includes nine Base RefoldingBuffers and seven additional buffer additives. The Base RefoldingBuffers form a matrix that includes a range of strong and weakdenaturant conditions for the suppression of protein aggregation(Table 11). The supplied additives are used as additional matrixfactors, depending on the protein type being refolded (Table 11 andTable 12). Buffer components are examined at three concentrationlevels, allowing a wide spectrum of folding conditions to be testedwithin one experiment. The adjustable design allows matrix conditionsto be tailored to the target protein, preventing sample waste andunnecessary analysis, while maximizing refolding yields.

The Pro-Matrix™ Protein Refolding Kit is accompanied by a compre-hensive Refolding Guide with details on isolating, solubilizing andpurifying inclusion bodies; optimizing refolding conditions; andanalyzing refolding yields.

Figure 41. Set-up and handling of the Pro-Matrix™ Protein Refolding Kit.

Table 11. Formulation and matrix design of Pro-Matrix™ Protein Refolding Kit.

BaseRefolding Factor 1 Factor 2 Factor 3 Factor 4 Buffer* Guanidine**(M) L-Arginine (M) Additive 1*** Additive 2***

1 0 0 1 12 0 0.44 2 23 0 0.88 3 34 0.55 0 2 35 0.55 0.44 3 16 0.55 0.88 1 27 1.1 0 3 28 1.1 0.44 1 39 1.1 0.88 2 1

* Each Base Refolding Buffer is supplied as a 1.1X stock solution. Each buffer stockcontains the indicated denaturant concentrations as well as 55 mM Tris, 21 mM NaCl,0.88 mM KCl adjusted to pH 8.2.

** Addition of solubilized protein sample will supply additional guanidine (GdnHCl).*** Factors 3 and 4 are defined by the researcher for customization of matrix conditions

with the supplied additives (see Table). The numbers in the table refer to concentrationlevels that are specific for each additive.

Table 12. Buffer additives.

Buffer Additive Amount Buffer Additive AmountPolyethylene Glycol (PEG) (10 mM) 1 ml EDTA (100 mM) 1 mlDithiothreitol (DTT) 100 mg Divalent Cation Solution 0.5 mlReduced Glutathione (GSH) 120 mg NaCl (5 M) 1 mlOxidized Glutathione (GSSG) 100 mg

Ordering InformationProduct # Description Pkg. Size89867 Pro-Matrix™ Protein Refolding Kit 100

Contains sufficient components to conduct Reactions/Kit100 refolding experiments (1 ml each).Includes: Base Refolding Buffers 1-9

1 (55 mM Tris, 21 mM NaCl, 0.88 mM KCl, pH 8.2) 10 ml2 (440 mM L-arginine, 55 mM Tris, 10 ml

21 mM NaCl, 0.88 mM KCl, pH 8.2)3 (880 mM L-arginine, 55 mM Tris, 10 ml

21 mM NaCl, 0.88 mM KCl, pH 8.2)4 (550 mM guanidine, 55 mM Tris, 10 ml

21 mM NaCl, 0.88 mM KCl, pH 8.2)5 (550 mM guanidine, 440 mM L-arginine, 10 ml

55 mM Tris, 21 mM NaCl, 0.88 mM KCl, pH 8.2)6 (550 mM guanidine, 880 mM L-arginine, 10 ml

55 mM, Tris, 21 mM NaCl, 0.88 mM KCl, pH 8.2)7 (1.1 M guanidine, 55 mM Tris, 21 mM 10 ml

NaCl, 0.88 mM KCl, pH 8.2)8 (1.1 M guanidine, 440 mM L-arginine, 10 ml

55 mM Tris, 21 mM NaCl, 0.88 mM KCl, pH 8.2)9 (1.1 M guanidine, 880 mM L-arginine, 10 ml

55 mM Tris, 21 mM NaCl, 0.88 mM KCl, pH 8.2)Dithiothreitol (DTT) 100 mgReduced Glutathione (GSH) 120 mgOxidized Glutathione (GSSG) 100 mg10 mM Polyethylene Glycol (PEG) 1 mlDivalent Cation Solution 0.5 ml 5 M NaCl 1 ml100 mM EDTA 1 mlRefolding Guidebook

Isolate and wash inclusion bodies.

Solubilize and reduce inclusion bodies.

Add 50 µl solubilized and reduced inclusion bodies to each of the nine tubes.

Add supplied additives as required for target protein to the above nine tubes. Adjust volume to 950 µl.

Dispense 900 µl of the Base Refolding Buffers in nine tubes.

Incubate overnight. Analyze for refolding.


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Protein Refolding

Protein RefoldingProtease Inhibitors

Protein Desa

lting

Inclusion Body Solubilization ReagentEffectively solubilizes insoluble protein.

Highlights:• Achieves the essential first step for subsequent insoluble protein

refolding procedures• One easy-to-use solution

Bacteria are widely used for recombinant protein expression.However, 70-80% of proteins produced by recombinant techniquesin E. coli form inclusion bodies (i.e., protein aggregates). Once theseaggregates are formed, it is very difficult to solubilize them. PierceInclusion Body Solubilization Reagent is designed to retrieve expressedprotein in soluble form after lysis and extraction procedures.

A proprietary denaturant contained in this reagent provides the mosteffective means for solubilizing aggregated proteins. Additionalcomponents, such as a reducing agent and a chelating agent, maybe added to the reagent, depending on the particular application.The following protocol is a generally applicable method useful inmoving protein from the insoluble inclusion body state into solutionin a gentle, phased approach. This is the first and essential stepbefore proceeding with insoluble protein refolding procedures.(See ProMatrix™ Protein Refolding Kit, page 33.)

Inclusion body purification. Recombinant proteins expressed inbacteria often form inclusion bodies, especially when they areexpressed at high levels. It is not known exactly how they are formed,but it is thought that the protein within the inclusion body is partiallyor incorrectly folded. The advantage of inclusion bodies is thatthey generally allow greater levels of expression, and they can beeasily separated from a large proportion of bacterial cytoplasmicproteins by centrifugation, giving an effective purification step.B-PER® Reagent and its companion protocol have been optimizedfor inclusion body purification after soluble protein extraction.1Although other methods may also be used, optimization is requiredto obtain inclusion body with good yield and purity.

Protein refolding. Because this reagent contains denaturant, theprotein is denatured after solubilization. To obtain active protein, itis necessary to perform protein refolding. Several methods havebeen published describing protein refolding.2-4 A suggested protocolis provided for proceeding with refolding procedures. Because everyprotein possesses unique folding properties, the optimal refoldingprotocol for any given protein must be empirically determined.

Compatibility. The denaturant included in the Inclusion BodySolubilization Reagent will precipitate in SDS-PAGE sample buffer.However, this reagent is compatible with Pierce Coomassie Plus –The Better Bradford™ Protein Assay Reagent (Product # 23236).The denaturant can be removed by dialysis before performingSDS-PAGE analysis and other protein assays.

References1. Chu, R., Mallia, A.K., Brennan, T. and Klenk, D. (1998). Recombinant protein extraction

from E. coli using B-PER® Bacterial Protein Extraction Reagent. Previews 2(1), 12-13.2. Mukhopadhyay, A. (1997). Inclusion bodies and purification of protein in biologically

active forms. Adv. Biochem. Eng. Biotechnol. 56, 61-109.3. Rudolph, R. and Lilie, H. (1996). In-vitro folding of inclusion body proteins. FASEB J.

10, 49-56.4. Marston, F.A.O. and Hartley, D.L. (1990). Solubilization of protein aggregates. Meth.

Enzymol. 182, 264-276.

Example Solubilization Protocol1. Purify the inclusion body using B-PER® Bacterial Protein Extraction Reagent

(Product # 78248). Be sure to follow the manufacturer-supplied instructions.Inclusion bodies purified by other methods are also applicable to the followingprotocols. The purity of the inclusion body prep may be analyzed by SDS-PAGEbefore performing the solubilization procedures. The purity of the inclusionbody prep will not affect the solubilization efficiency. However, if a subsequentrefolding procedure is desired, a greater than 90% purity is preferred.

2. Estimate the amount of inclusion body prep by subtracting the weight of the wetinclusion body pellet contained in a centrifuge tube from the weight of the cen-trifuge tube. Use 8 ml of the Inclusion Body Solubilization Reagent for 1 g ofwet inclusion body pellet. More reagent may be used to dilute the protein further.

3. Resuspend the pellet in an appropriate amount of the Inclusion BodySolubilization Reagent by either vigorous vortex mixing or by pipetting untilsuspension is homogeneous. Shake the suspension for 30 minutes.

4. Remove cell debris by centrifugation at 27,000 x g (15,000 rpm for BeckmanJA20 rotor) for 15 minutes.

5. Collect the supernatant that contains the solubilized protein from the inclusionbody. If a protein assay is desired, Pierce Coomassie Plus – The BetterBradford™ Protein Assay Reagent (Product # 23236) is compatible. ForSDS-PAGE analysis, it is necessary to remove the denaturant contained inthe solubilization reagent by dialysis.

Protein Refolding Using Dialysis MethodMaterials required: • B-PER® Bacterial Protein Extraction Reagent: For inclusion

body purification• Inclusion Body Solubilization Reagent: For inclusion

body solubilization • DTT: Reducing agent (Product # 20290)• Urea: Mild denaturant• Slide-A-Lyzer® Dialysis Cassette: Dialysis (Product # 66410)

Suggested Refolding Protocol1. Purify inclusion body from bacteria using B-PER® Bacterial Protein Extraction

Reagent and solubilize inclusion body protein using Inclusion Body SolubilizationReagent following respective protocols. If disulfide bonds are involved inrefolding, add DTT to 5 mM to the Inclusion Body Solubilization Reagentduring the solubilization step.

2. Prepare 1 liter of 6 M Urea solution in a 3.5 liter glass or plastic beaker.3. Use an 18-gauge needle and a 10 ml syringe to transfer 8 ml of the Inclusion

Body Protein Solution to a 3-15 ml Slide-A-Lyzer® Cassette. Dialyze theinclusion body protein against 6 M Urea for 6 hours.

4. Add 250 ml of 25 mM Tris•HCl (pH 7.5) to the beaker every 6-12 hours.Once the volume reaches 3 liters, replace the dialysis solution with 2 litersof 25 mM Tris•HCl (pH 7.5) and 150 mM NaCl. Dialyze for another 6 hours.To maintain protein stability, perform the dialysis in a cold room (4-8˚C).Precipitation may form during dialysis; however, there will always be someprotein remaining in solution.

5. Recover the sample from the dialysis cassette using a similar-sized needleand syringe. Remove any visible insoluble material by centrifugation.Determine the folding status of the remaining soluble protein by activityassay or other methods.

Ordering InformationProduct # Description Pkg. Size78115 Inclusion Body Solubilization Reagent 100 ml

Highlights:• Exceptional protein recovery• Wide product offering accommodates your sample needs• Easy-to-use with no cumbersome column preparation

or equilibration • No screening fractions for protein or waiting for protein to

emerge by gravity flow• Minimal sample dilution

Although numerous techniques and resins for desalting are available,most have many drawbacks, including significant sample loss,long processing times and the need to collect multiple fractions.Zeba™ Desalt Spin Columns provide excellent protein recoverywithout the limitations associated with other desalting methods.With the introduction of Zeba™ Desalt Spin Columns in 2, 5 and10 ml formats to complement the Micro and 0.5 ml versions, theZeba™ Desalt Spin Column family of products allows processingof samples volumes ranging from 2 µl to 4 ml (Table 13).

Table 13. Recommended sample volumes for Zeba™ Spin Columns.

Resin Bed Sample Volume75 µl (micro) column 2-12 µl

0.5 ml column 30-130 µl2 ml column 200-700 µl 5 ml column 600-2,000 µl10 ml column 1,500-4,000 µl

The easy-to-use Zeba™ Spin-Column Format dramatically improvesresults over standard drip-column methodologies, eliminating the need to wait for samples to emerge by gravity flow and theneed to monitor fractions for protein recovery. Zeba™ DesaltingColumns require no chromatographic system, cumbersome columnpreparation or equilibration and they can process multiple samplesin ~8 minutes.

Zeba™ Desalt Spin Columns contain a proprietary high-performancedesalting resin, exclusive to Pierce, that offers exceptional desaltingand protein-recovery characteristics compared to other commerciallyavailable resins (Figure 41). Samples containing as low as 25 µg/mlof protein can be processed, providing exceptional protein recoveryand ≥ 95% retention of salts and other small molecules (< 1,000 MW).

Figure 41. Increased protein recovery with Zeba™ Desalt Spin Columns.Samples of bovine serum albumin (BSA) at Figure 41A. 250 µg/ml and Figure 41B.25 µg/ml in 1 M NaCl were desalted with the 2 ml Zeba™ Desalt Spin Columnsand other commercial desalting resins using similar formats. A portion of therecovered sample (10 µl) was analyzed by SDS-PAGE. The remaining samplewas used for conductivity measurements and BCA™ Protein Assay (Product# 23225) to determine protein concentration. Zeba™ Desalting Resin providessignificantly greater protein recovery under all conditions tested. Conductivityand protein recovery values after desalting are indicated for 250 µg/ml samples.

Ordering InformationProduct # Description Pkg. Size89877 Zeba™ Micro Desalt Spin Columns† 25/pack89878 Zeba™ Micro Desalt Spin Columns† 50/pack89882 Zeba™ Desalt Spin Columns, 0.5 ml 25/pack89883 Zeba™ Desalt Spin Columns, 0.5 ml 50/pack89889 Zeba™ Desalt Spin Columns, 2 ml 5/pack89890 Zeba™ Desalt Spin Columns, 2 ml 25/pack89891 Zeba™ Desalt Spin Columns, 5 ml 5/pack89892 Zeba™ Desalt Spin Columns, 5 ml 25/pack89893 Zeba™ Desalt Spin Columns, 10 ml 5/pack89894 Zeba™ Desalt Spin Columns, 10 ml 25/pack

Empty Zeba Spin Columns

89879 Zeba™ Micro Spin Empty Columns† 50/pack89896 Zeba™ Empty Spin Columns, 2 ml 25/pack89897 Zeba™ Empty Spin Columns, 5 ml 25/pack89898 Zeba™ Empty Spin Columns, 10 ml 25/pack† U.S. patent pending on Zeba™ Micro Column Technology.

Sample Size700 µl

Sample Size200 µl + 50 µl stacker

Cont

rol

5.2

Zeba

Bra

nd

97%

0.19

Com

p. B

78%

0.45

Com

p. G

79%

0.73

Sample 250 µg/ml BSA (66 kDa)

Conductivity (mho)

Recovery

Sample 25 µg/ml BSA (66 kDa)

Cont

rol

5.2

Zeba

Bra

nd

90%

0.14

Com

p. B

56%

0.07

Com

p. G

29%

0.14

A.

B.

For Technical or Customer Assistance, Call 800-874-3723 or Fax 800-842-5007 35www.piercenet.com/pop95d • E-mail Customer Service: [email protected]

Prot

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Zeba™ Desalt SpinColu

mns

Zeba™ Desalt Spin ColumnsDesalt sample volumes ranging from 2 µl to 4 ml with Zeba™ Desalt Spin Columns and experience exceptional protein recovery quickly.

G r a s p t h e P r o t e o m e ™

Protein Concentration

Concentrate for SuccessiCON™ Concentrators are advanced disposable

ultrafiltration centrifugal devices for concentration and diafiltration/buffer-exchange of biological samples such as enzymes, antigens or antibodies. The innovative conical design* and high-performance regenerated cellulose membrane provide excellent protein concentration and recovery from dilute protein samples. iCON™ Concentrators are available with MWCO of 9K and 20K in 7 and 20 ml volume sizes.

Highlights:• Achieve 150- to 400-fold protein concentrations in less than 30 minutes• Accommodate concentration volumes over a wide working range

(7 ml = 1-7 ml and 20 ml = 5-20 ml ranges)• Desalt and exchange buffers• Uses the maximum membrane surface area, providing unsurpassed

protein concentration• Compatible with swinging-bucket or fixed-angle rotors• No invert spin required• Excellent recovery of dilute proteins

Ordering InformationProduct # Description Pkg. Size89884 iCON™ Concentrators, 7 ml, 9K MWCO 25/pkg.89885 iCON™ Concentrators, 20 ml, 9K MWCO 25/pkg.89886 iCON™ Concentrators, 7 ml, 20K MWCO 25/pkg.89887 iCON™ Concentrators, 20 ml, 20K MWCO 25/pkg.

www.piercenet.com/icon

High-Performance Dialysis


The new 30 ml (working range12-30 ml) Slide-A-Lyzer ® DialysisCassette* was built for dialysis ina BIG way. It comes with its owninternal buoy.The color-coded transparentframes of the Slide-A-Lyzer ®

Cassette Family allow you toinstantly know the molecularweight cut-off of the membraneand view the placement of yourneedle during sample injection.

BigBuoy™

CassetteOur line keeps getting BIGger and better.

Highlights:• The 30 ml cassette has the

buoy built into its frame• > 95% sample recovery• No knots, caps, lids or clamps

to loosen, fall off, open or leak• A rigid frame that permits

smooth and complete withdrawal of samples

• Color-coded frames indicate membrane MWCO

To view demos, see a completelist of products or to downloadan instruction booklet, visitwww.piercenet.com/dialysis.

Protein Refolding


www.piercenet.com/pop95dA comprehensive Refolding Guide isincluded in your Pro-Matrix™ ProteinRefolding Kit. It includes details on isolating,solubilizing and purifying inclusion bodies;optimizing refolding conditions; and analyzing refolding yields.

• High-yield refolding conditions – reagents and conditionslimited to those proven effective for high-concentrationprotein refolding experiments

• Adjustable three-level matrix format – customizesrefolding experiments to specific protein, reduces optimization time and simplifies data interpretation

• High-purity reagents – offers quality reagents formulatedusing stringent standards so that consistent results are attained

Product # Description89867 Pro-Matrix™ Protein Refolding Kit

Bring your protein back into the fold. Restore native protein function with the Pro-Matrix™ Protein Refolding Kit.

The Pro-Matrix™ Protein Refolding Kit is an enabling tool for determining and optimizing conditions for restoring native protein function from inclusion bodies.

Tel: 815-968-0747 or 800-874-3723 • Fax: 815-968-7316 • Technical Assistance E-mail: [email protected] • Customer Assistance E-mail: [email protected] the United States, visit our web site or call 815-968-0747 to locate your local Perbio Science branch office (below) or distributor

© Pierce Biotechnology, Inc., 2005. Printed in the U.S.A. Pierce products are supplied for laboratory or manufacturing applications only.All-In-One™, BCA™, BigBuoy™, B-PER®, Brij®, BupH™, CL-XPosure™, Coomassie Plus – The Better Bradford™, Extracti-Gel®, GelCode™, Halt™, ImmunoPure®, LightShift®, Lyse-N-Go™, Mem-PER®, M-PER®, NE-PER®, PAGEprep®,

Poppers™, Pro-Matrix™, SDS-Out™, Slide-A-Lyzer® , SnakeSkin®, SuperBlock® , SuperSignal®, Surfact-Amps®, Surfact-Pak™, T-PER®, Y-DER™, Y-PER® and Zeba™ are trademarks of Pierce Biotechnology, Inc.

Polytron® is a trademark of Kinematica AG Company. Sonifier® is a registered trademark of Branson.Teflon® is a trademark of E.I. du Pont de Nemours & Co., Inc. Triton® is a registered trademark of Rohm & Haas Company. Tween® is a trademark of ICI Americas. Waring® is a trademark of Dynamics Corporation of America.

U.S. patent pending on Mitochondria Isolation Kit Technology and Zeba™ Micro Column Technology. B-PER® Technology is protected by U.S. patent # 6,174,704. BCA™ Technology is protected by U.S. patent # 4,839,295. Slide-A-Lyzer® MINI Dialysis Unit Technology is protected by U.S. patent # 6,039,871. SuperSignal® Technology is protected

by U.S. patent # 6,432,662. PCR is covered by U.S. patent #’s 4,683,195 and 4,683,202, which are owned by Hoffmann-La Roche, Inc. Use of the PCR process requires a license.

Belgium & Dist.:Tel +32 53 85 7184

[email protected]

China:Tel +86 10 8049 [email protected]

France:Tel 0800 50 82 15

[email protected]

Germany: Tel 0228 9125650

[email protected]

Hong Kong:Tel 852 2753 0686

[email protected]

The Netherlands: Tel 076 50 31 880

[email protected]

United Kingdom: Tel 0800 252185

[email protected]

Switzerland: Tel 0800 56 31 40

[email protected]

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