2. Development of Western Blots for Actin without the use of radioactivity Geoff Theobald STEP Summer Internship Program June 2003

3. Objective To create a Western Blot without the use of radioactivity.

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

5. Background Information Actin: Is the protein we detected using a Western Blot. Has a molecular weight of 42,000-43,000 daltons. Works with myosin (another protein) in muscle contraction. Is also involved in the structure in most cells.

6. Methods Polyacrylamide gel electrophoresis Western Blot Probing with an antibody to actin Detection by fluorescence

7. Polyacrylamide gel electrophoresis Separates proteins by size Protein standards are used to determine the size of Actin

8. Western Blot A western is when you transfer protein out of polyacrylamide gel and into a membrane.western A Western Blot is the result of the transfer.Western Blot Gel Pic. Of membrane

9. Probing with an antibody to actin Probe blot with antibody N-term C-term Actin Protein

10. Detection by fluorescence Key = Actin = Primary Antibody = Secondary Antibody = DDAO = DDAO phosphate = Alkaline phosphatase Actin Primary Rabbit Antibody for Actin Secondary Antibody for Rabbit Alkaline Phosphatase DDAO phosphate DDAO Western Blot

11. Results: detection with UV light There was no clear signal. Pic. of Blot exposed to UV light

12. Fluorescent dye can be visualized with UV or white light Advantages UV: we can distinguish red light White light: stronger excitation

13. Absorption/emission spectrum of DDAO Use white light with a blue filter

14. Results: detection with white light There was no clear signal. Pic. Of blot exposed to white light

15. Chemiluminescence assay Worked well. Continued experiments with this assay. Pic of dot blot with all actin antibody Pic of membrane with all actin antibody

16. Conclusions 1.Detection of fluorescence with UV light did not work. 2.Detection of fluorescence with white light and a blue filter did not work. 3.Since fluorescence assay did not work well, but chemiluminescence worked, we will concentrate on that assay.

17. Acknowledgements Dr. Guzman, Biology Dept. Dr. Metz, Biology Dept. Mrs. Bloom, STEP program I would like to thank each and everyone of you for all of your advice, support, and fun I had these past two weeks.