1 / 18

By Emily Johnson Summer STEP Program, 2003

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

avedis
Download Presentation

By Emily Johnson Summer STEP Program, 2003

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Development of a Western Blot For Actin Using Chemiluminescence By Emily Johnson Summer STEP Program, 2003

  2. Objective:• Develop Non-radioactive Western Specific Aims: • Show we can use the actin antibody to specifically recognize actin • 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

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

  4. cytoplasm nucleus DNA Transcription Translation RNA RNA Protein Background Information GeneExpression

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

  6. Methods www.sdsc.edu/Publications/ScienceAlive/ A Picture of Protein

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

  8. Western Blot • Transferring from a gel to a membrane

  9. Probing • Antibody sticks specifically to actin

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

  11. Secondary Antibody-HRP Peracid substrate Actin X-ray Film Membrane Product + Light Primary Antibody Principles of Chemiluminescence

  12. www.nhlbi.nih.gov/ labs/cellbiology/ A picture of Actin in action! Results

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

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

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

  16. Conclusions • 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 antibodies to certain parts of the actin do specifically recognize actin.

  17. Acknowledgements • Dr. Guzman-Biology Department • Mrs. Bloom-STEP Coordinator • Dr. Metz-Biology Department • Campbell University • 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.

  18. The Western!!!

More Related