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Development of Western Blots for Actin without the use of radioactivity

Development of Western Blots for Actin without the use of radioactivity. Geoff Theobald STEP Summer Internship Program June 2003. Objective. To create a Western Blot without the use of radioactivity. Significance. Safety & disposal

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Development of Western Blots for Actin without the use of radioactivity

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

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

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

  4. 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.

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

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

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

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

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

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

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

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

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

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

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

  16. 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.

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