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Western Blot

Western Blot. Megan Sova Stacie Huffmon Beth Meyer. What is the method?. This is the last step of a long process to be able to identify a specific protein. How is it performed?. Obtain a protein sample Run samples on gel to separate for size (SDS-PAGE) Transfer gel proteins onto membrane

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Western Blot

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  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? • Obtain a protein sample • Run samples on gel to separate for size (SDS-PAGE) • Transfer gel proteins onto membrane • Wash antibody over surface to select protein of interest • 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/genomics/method/Westernblot.html • http://www.molecularstation.com/protein/western-blot/ • http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&blobtype=pdf&artid=1134099 • Dr. Baker

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