by emily johnson summer step program, 2003
DESCRIPTION
Development of a Western Blot For Actin Using Chemiluminescence. By Emily Johnson Summer STEP Program, 2003. Objective: • Develop Non-radioactive Western. Specific Aims: Show we can use the actin antibody to specifically recognize actin - PowerPoint PPT PresentationTRANSCRIPT
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By Emily JohnsonBy Emily Johnson
Summer STEP Program, Summer STEP Program, 20032003
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Objective:Objective: • • Develop Non-radioactive Develop Non-radioactive WesternWestern
Specific Aims:Specific Aims: Show we can use the actin antibody Show we can use the actin antibody
to specifically recognize actinto specifically recognize actin See if the antibody will recognize the See if the antibody will recognize the
protein produced in a test tubeprotein produced in a test tube Show that antibodies to certain parts Show that antibodies to certain parts
of the actin protein will also of the actin protein will also specifically recognize actinspecifically recognize actin
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SignificanceSignificance
Safety & disposalSafety & disposal Use in college lab: students learn about gene Use in college lab: students learn about gene
expression and molecular biologyexpression and molecular biology
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Background InformationBackground InformationGeneGene ExpressionExpression
cytoplasmnucleus
DNA
RNATranscription
RNA ProteinTranslation
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Antibodies That Detect Actin…Antibodies That Detect Actin…
Key:
= antibody to entire actin
= antibody to N-terminus of actin
= antibody to C-terminus of actin
N-termC-term
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MethodsMethods
www.sdsc.edu/Publications/ScienceAlive/
A Picture of Protein
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Polyacrylamide Polyacrylamide Gel Gel ElectrophoresisElectrophoresis
•Process of separating the protein by size
•Protein standards are used to determine the size of actin
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Western BlotWestern Blot
•Transferring from a gel to a membrane
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ProbingProbing
Antibody sticks specifically to actinAntibody sticks specifically to actin
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Detection By Detection By ChemiluminescencChemiluminescencee
Flow Diagram:Flow Diagram:In the Dark Room
The Dark Room :)
Detection method complete!
Separate protein sample by Separate protein sample by electrophoresiselectrophoresis
Transfer to Transfer to membranemembrane
Block non-specific Block non-specific sitessites
Incubate in primary Incubate in primary antibodyantibody
Incubate in HRP-labeled Incubate in HRP-labeled conjugateconjugate
ECL detection ECL detection reagentsreagents
Expose to Expose to filmfilm
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Principles of Principles of ChemiluminescenceChemiluminescence
ActinActinPeracid Peracid substratesubstrate
Product + Product + LightLight
X-rayX-ray
FilmFilm
MembraneMembrane
Primary Antibody
Secondary Antibody-HRP
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ResultsResults
www.nhlbi.nih.gov/ labs/cellbiology/
A picture of Actin in action!
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Does the Actin Antibody Does the Actin Antibody Specifically Recognize Specifically Recognize Actin?Actin?
Binds to a band at ~42-Binds to a band at ~42-43kd43kd
Binds to the band seen with Binds to the band seen with pure actinpure actin
A little binding to markersA little binding to markers *Non-specific?*Non-specific?
Something similar?Something similar?
Binds to actin, but not Binds to actin, but not another protein called BSAanother protein called BSA
Uneven spreading of ECL Uneven spreading of ECL reagentreagent
Antibody=AllAntibody=All
BSA
cardiac
muscle
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Do Antibodies, to Certain Parts of Do Antibodies, to Certain Parts of the Actin, Specifically Recognize the Actin, Specifically Recognize Actin?Actin?
All 3 antibodies have low All 3 antibodies have low binding to BSAbinding to BSA
Unsure why,but , in general, Unsure why,but , in general, some dots don’t change with some dots don’t change with concentrationconcentration
*Technical?*Technical? Blots show strong signal of Blots show strong signal of
actin proteinactin proteinN-term Dot BlotN-term Dot Blot
All Actin Dot BlotAll Actin Dot Blot
C-term Dot BlotC-term Dot Blot
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Does the Antibody Recognize Does the Antibody Recognize the Protein Produced in a the Protein Produced in a Test Tube?Test Tube? Several bands bind each of Several bands bind each of
the three antibodiesthe three antibodies Binding IV, some of expected Binding IV, some of expected
size, but need control reaction size, but need control reaction without genewithout gene
IV IV 3 main bands 3 main bands
Interesting is smallerInteresting is smaller
Band not bound by Band not bound by
C-term antibodyC-term antibody
N-term gelN-term gel
All Actin All Actin GelGel
C-term gelC-term gel
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ConclusionsConclusions
The actin antibody to the entire actin The actin antibody to the entire actin specifically recognizes actin.specifically recognizes actin.
The antibody seems to recognize actin The antibody seems to recognize actin produced in the test tube. More produced in the test tube. More experiments are necessary.experiments are necessary.
The antibodies to certain parts of the The antibodies to certain parts of the actin do specifically recognize actin.actin do specifically recognize actin.
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AcknowledgementsAcknowledgements
• Dr. Guzman-Biology DepartmentDr. Guzman-Biology Department• Mrs. Bloom-STEP CoordinatorMrs. Bloom-STEP Coordinator• Dr. Metz-Biology DepartmentDr. Metz-Biology Department• Campbell UniversityCampbell University• All the people who created the STEP All the people who created the STEP
programprogram
Thank you for all your help with our Thank you for all your help with our experiments and presentations. experiments and presentations. Without you none of this would be Without you none of this would be possible for us.possible for us.
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