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CHAPTER 9/EXERCISE 9 WESTERN BLOTTING AND DETECTION OF SPECIFIC PROTEINS ON NITROCELLULOSE Western Blotting : detection of specific individual protein from amongst the hundreds or thousands of different proteins on an electroblotted membrane o Antibodies : the probes that are used to identify the protein of interest on an electroblotted membrane o Uses a 2 antibody system : one antibody binds to protein of interest, 2 nd antibody binds to 1 st antibody and also carries a detectable label to identify location of the protein of interest Using Antibodies to locate specific protein on an electrotransfer membrane o We are testing liver and tripe protein crude extracts for the presence of albumin protein o Albumin : synthesized in the liver and secreted into the bloodstream where they perform a vital function in exchanging fluids, gases, and wastes between capillaries and tissues They also bind and transport many molecules through the circulatory system Are osmotically active solutes Maintain a high solute concentration in the capillaries, which causes the fluids to return to the capillaries by osmosis o Use Western blotting ( immunoblotting) to identify a protein with a labeled antibody BLOCKING THE ELECTROBLOTTED MEMBRANE: o Before antibodies can be applied to electroblotted nitrocellulose membrane, membrane must be treated with Blocking Solution o Blocking Solution: composed of protein (non-fat milk) that covers sites on the membrane to which no protein had previously been bound during electroblotting This allows antibodies to not be trapped and bound to the membrane directly The antibodies will not get trapped on the nitrocellulose surface, but will only bind to specific antigens o Primary antibody Solution: one that binds directly to the target protein Anti-albumin ultimately indicate which protein band or bands on the nitrocellulose contain albumin protein Albumin antibody recognizes albumin protein The full name includes the animal in which it was raised (ex: rabbit anti-albumin antibody) Nitrocellulose membrane is incubated overnight in primary antibody solution and
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This note was uploaded on 03/20/2008 for the course BIO 205L taught by Professor Hanson during the Fall '07 term at University of Texas.

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