BIS 104 PQ 19 Spring 2010

BIS 104 PQ 19 Spring 2010 - are linked to the heavy chains...

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BIS PQ 19 Spring 2010 19. Let’s suppose that you wish to produce a polyclonal antibody against the human glycoprotein, PECAM-1 to be used in IHC and western blotting experiments. You carry out the following steps: 1. PECAM-1 glycoprotein is purified from the PMs of human ECs. 2. PECAM-1 is injected into a rabbit several times over several weeks time. As a control, a sample of pre-immune serum is collected from your rabbit. 3. The IgG protein fraction is purified from the rabbit serum by a combination of gel exclusion chromatography and ion exchange chromatography. The IgG molecule has a mol wt of 150kDa and is a tetramer composed of 2 identical “light” chains (mol wt = 25kDa each) and 2 identical “heavy” chains (mol wt = 50kDa each). The heavy chains are linked to each other by intermolecular disulfide bonds and the light chains
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Unformatted text preview: are linked to the heavy chains by intermolecular disulfide binds. To evaluate the purity of your IgG preparation, you perform the following tests: A sample of your IgG prep is run on SDS-PAGE under reducing conditions. A sample of your IgG prep is run on SDS-PAGE under non-reducing conditions. Both gels are stained with Coomassie Blue (CB) and the Rm of each stained band is estimated by comparison with a standard curve of proteins with known mol wts. a. Diagram the gel lanes and CB banding patterns you would expect to see from a pure IgG prep. b. Describe how you could use SDS-PAGE and western blotting to verify the specificity of your anti-PECAM antibody. c. Describe how you would use a sample of the rabbit pre-immune serum as a control....
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This note was uploaded on 04/06/2011 for the course BIS 104 taught by Professor Scholey during the Spring '08 term at UC Davis.

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BIS 104 PQ 19 Spring 2010 - are linked to the heavy chains...

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