Western Blot Megan Sova Stacie Huffmon Beth Meyer.

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Presentation transcript:

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? 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

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

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

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.

References enomics/method/Westernblot.htmlhttp:// enomics/method/Westernblot.html estern-blot/ estern-blot/ er.fcgi?tool=pmcentrez&blobtype=pdf&arti d= http:// er.fcgi?tool=pmcentrez&blobtype=pdf&arti d= Dr. Baker