By Emily JohnsonBy Emily Johnson

Summer STEP Program, Summer STEP Program, 20032003

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

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

Background InformationBackground InformationGeneGene ExpressionExpression

cytoplasmnucleus

DNA

RNATranscription

RNA ProteinTranslation

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

MethodsMethods

www.sdsc.edu/Publications/ScienceAlive/

A Picture of Protein

Polyacrylamide Polyacrylamide Gel Gel ElectrophoresisElectrophoresis

•Process of separating the protein by size

•Protein standards are used to determine the size of actin

Western BlotWestern Blot

•Transferring from a gel to a membrane

ProbingProbing

Antibody sticks specifically to actinAntibody sticks specifically to actin

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

Principles of Principles of ChemiluminescenceChemiluminescence

ActinActinPeracid Peracid substratesubstrate

Product + Product + LightLight

X-rayX-ray

FilmFilm

MembraneMembrane

Primary Antibody

Secondary Antibody-HRP

ResultsResults

www.nhlbi.nih.gov/ labs/cellbiology/

A picture of Actin in action!

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

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

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

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.

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.