Discussion Section 2 - Add SDS + -mercaptoethanol Problem 1...

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Add SDS + β-mercaptoethanol
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Problem 1 Problem 1) Hsp70 molecular chaperones are thought to bind to hydrophobic regions of nascent polypeptides on ribosomes. The bacteria E. coli has an Hsp70 chaperone called DnaK. To test whether DnaK binds to polypeptides as soon as they are synthesized you use a pulse-chase and immunoprecipitation analysis. You do the following experiments: A) You obtained from another lab antibodies that supposedly bind specifically to DnaK but they did not show you the supporting evidence. Your first task is to test whether you can use these antibodies to immunoprecipitate DnaK and any proteins bound to DnaK. You also need to show that this co- immunoprecipitation is specific, that is, that any protein that shows up in the gel was truly bound to DnaK instead of being bound by the antibodies or the beads themselves. (Some antibody or bead preparations are very sticky and will easily bind random proteins. You do not want this!) You fed E. coli bacteria a 1 minute pulse of 35S-methionine without any
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This note was uploaded on 02/27/2011 for the course BIO 320 taught by Professor Staff during the Spring '08 term at University of Texas at Austin.

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Discussion Section 2 - Add SDS + -mercaptoethanol Problem 1...

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