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 Show we can use the actin antibody to specifically recognize actin See if the antibody will recognize the protein produced in a test tube See if the antibody will recognize the protein produced in a test tube Show that antibodies to certain parts of the actin protein will also specifically recognize actin Show that antibodies to certain parts of the actin protein will also specifically recognize actin

Significance Safety & disposal Safety & disposal Use in college lab: students learn about gene expression and molecular biology Use in college lab: students learn about gene expression and molecular biology

Background Information Gene Expression cytoplasm nucleus DNA RNA Transcription RNA Protein Translation

Antibodies That Detect Actin… Key: = antibody to entire actin = antibody to N-terminus of actin = antibody to C-terminus of actin N-term C-term

Methods A Picture of Protein

Polyacrylamide Gel Electrophoresis Process of separating the protein by size Protein standards are used to determine the size of actin

Western Blot Transferring from a gel to a membrane

Probing Antibody sticks specifically to actin Antibody sticks specifically to actin

Detection By Chemiluminescence Flow Diagram: In the Dark Room The Dark Room :) Detection method complete! Separate protein sample by electrophoresis Transfer to membrane Block non-specific sites Incubate in primary antibody Incubate in HRP-labeled conjugate ECL detection reagents Expose to film

Principles of Chemiluminescence Actin Peracid substrate Product + Light X-rayFilm Membrane Primary Antibody Secondary Antibody-HRP

Results labs/cellbiology/ A picture of Actin in action!

Does the Actin Antibody Specifically Recognize Actin? Binds to a band at ~42-43kd Binds to a band at ~42-43kd Binds to the band seen with pure actin Binds to the band seen with pure actin A little binding to markers A little binding to markers *Non-specific? *Non-specific? Something similar? Something similar?  Binds to actin, but not another protein called BSA Uneven spreading of ECL reagent Uneven spreading of ECL reagentAntibody=All BSA cardiac muscle

Do Antibodies, to Certain Parts of the Actin, Specifically Recognize Actin? All 3 antibodies have low binding to BSA All 3 antibodies have low binding to BSA Unsure why,but, in general, some dots don’t change with concentration Unsure why,but, in general, some dots don’t change with concentration*Technical?  Blots show strong signal of actin protein N-term Dot Blot All Actin Dot Blot C-term Dot Blot

Does the Antibody Recognize the Protein Produced in a Test Tube? Several bands bind each of the three antibodies Several bands bind each of the three antibodies Binding IV, some of expected size, but need control reaction without gene Binding IV, some of expected size, but need control reaction without gene IV  3 main bands IV  3 main bands Interesting is smaller Band not bound by Band not bound by C-term antibody N-term gel All Actin Gel All Actin Gel C-term gel

Conclusions The actin antibody to the entire actin specifically recognizes actin. The actin antibody to the entire actin specifically recognizes actin. The antibody seems to recognize actin produced in the test tube. More experiments are necessary. The antibody seems to recognize actin produced in the test tube. More experiments are necessary. The antibodies to certain parts of the actin do specifically recognize actin. The antibodies to certain parts of the actin do specifically recognize actin.

Acknowledgements Dr. Guzman-Biology Department Dr. Guzman-Biology Department Mrs. Bloom-STEP Coordinator Mrs. Bloom-STEP Coordinator Dr. Metz-Biology Department Dr. Metz-Biology Department Campbell University Campbell University All the people who created the STEP program All the people who created the STEP program Thank you for all your help with our experiments and presentations. Without you none of this would be possible for us.