1. Western Blot Megan Sova Stacie Huffmon Beth Meyer

2. What is the method? This is the last step of a long process to be able to identify a specific protein.

3. How is it performed? 1.Obtain a protein sample 2.Run samples on gel to separate for size (SDS-PAGE) 3.Transfer gel proteins onto membrane 4.Wash antibody over surface to select protein of interest 5.Reveal and detect protein of interest by incubating it with a colorless substrate (enzyme) that converts to a colored product that can be seen

4. Why do this method? Can detect one protein in a mixture of proteins Can give information on size and the amount of expression of the specific protein by location and thickness of band

5. Typical Data Negative: No bands present Positive: Bands at either p31 OR p24 AND bands present at either gp160 OR gp120 Indeterminate: Bands present, but pattern does not meet criteria for positivity

6. Importance Once the protein has been identified by using Western Blot, it can be isolated on the SDS-PAGE gel. The protein can then be analyzed for sequencing and gene determination.

7. References http://www.bio.davidson.edu/COURSES/g enomics/method/Westernblot.htmlhttp://www.bio.davidson.edu/COURSES/g enomics/method/Westernblot.html http://www.molecularstation.com/protein/w estern-blot/http://www.molecularstation.com/protein/w estern-blot/ http://www.pubmedcentral.nih.gov/picrend er.fcgi?tool=pmcentrez&blobtype=pdf&arti d=1134099http://www.pubmedcentral.nih.gov/picrend er.fcgi?tool=pmcentrez&blobtype=pdf&arti d=1134099 Dr. Baker