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RECOMBINANT DNA SESSION 07: DEVELOPMENT OF THE BLOT BACKGROUND The experimental method used in today’s session is functionally equivalent to a Western blot in its detection methodology. Our purpose is to perform a Southern blot to detect a DNA probe hybridized to specific DNA sequences. The purpose of a Western blot is to detect specific proteins. For your training as a biologist, it is educational to consider how a Western blot is performed relative to a Southern blot. In our work, the digoxigenin-labeled DNA probe is like the protein on a regular Western blot membrane. No secondary antibody is needed. There is so much label in the probe that the anti-digoxigenin antibody can be labeled by alkaline phosphatase conjugation and used directly on the blot. Source of antibodies for blotting - where do antibodies for blotting come from? B lymphocytes of the immune system can produce antibodies to almost any known chemical substance. The substance to which the immune system responds is called the antigen. The antibodies used in developing our blot are directed against digoxigenin, a small molecule related to steroids. Small molecules are usually not very antigenic by themselves but if coupled to a larger molecule, the immune system can more easily recognize them and respond. The carrier molecule used is usually deliberately chosen to not be very antigenic (stimulatory) itself. As you will learn in biology, the immune system does not A learn @ to respond. By randomly combining parts of a stock set of antibody genes, the immune system randomly generates a set of B cells capable of responding to millions of different antigens before the animal ever sees any antigen. Those few B cells that happen to be created that can bind molecules that look like digoxigenin are stimulated to divide (to produce more like themselves). Some of the stimulated B cells begin producing large amounts of antibody GOALS To equilibrate the nylon filter in antibody binding buffer and block again with blocking solution to block any free sites for nonspecific absorption of the anti-digoxigenin-alkaline phosphatase conjugate
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This note was uploaded on 05/03/2008 for the course BIO 2322 taught by Professor Spotswood during the Spring '08 term at The University of Texas at San Antonio- San Antonio.

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