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 • Use in college lab: students learn about gene expression and molecular biology
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.
Methods • Polyacrylamide gel electrophoresis • Western Blot • Probing with an antibody to actin • Detection by fluorescence
Polyacrylamide gel electrophoresis • Separates proteins by size • Protein standards are used to determine the size of Actin
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
C-term N-term Actin Protein Probing with an antibody to actin Probe blot with antibody
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
Pic. of Blot exposed to UV light Results: detection with UV light • There was no clear signal.
Fluorescent dye can be visualized with UV or white light Advantages • UV: we can distinguish red light • White light: stronger excitation
Absorption/emission spectrum of DDAO • Use white light with a blue filter
Pic. Of blot exposed to white light Results: detection with white light • There was no clear signal.
Pic of dot blot with all actin antibody Pic of membrane with all actin antibody Chemiluminescence assay • Worked well. Continued experiments with this assay.
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.
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.