BIO320_HW2_2011answers - Name _ BIO320 HW-2 Chaperones,...

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Name _____________________________________ BIO320 HW-2 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 35 S-methionine without any chase time. You lysed them in the absence of ATP, and then immunoprecipitated (IP) with the antibodies against DnaK. The IP was denatured in SDS sample buffer, run on an SDS-PAGE, and the radioactive bands were visualized with an x-ray film. You can see in Figure 1A, lane 1 that a protein the size of DnaK was immunoprecipitated along with a collection of many other labeled proteins. Given that you are looking at Radioactive proteins on the film, explain how is it possible that you are finding radioactive-labeled DnaK on your gel? This observation shows that some molecules of DnaK protein were synthesized during the pulse period. Those DnaK molecules that were made at this time ARE radioactive and will be visible on the film after immunoprecipitation. Of course, the cell already had many more molecules of DnaK that are NOT radioactive. These molecules are also immunoprecipitated and are present in the SDS-gel, but they will not contribute to the image on the film. B) You have a mutant strain of E. coli that does not have any DnaK. As you may expect, this strain cannot survive a heat shock but it can grow at lower temperatures. If you do the same Pulse labeling of these mutant cells, and immunoprecipitation with the DnaK antibodies you obtain the results shown in Figure 1A, lane 4. As you can see, there are NO protein bands visible on your film. What can you conclude about the co-immunoprecipitation of many proteins
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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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BIO320_HW2_2011answers - Name _ BIO320 HW-2 Chaperones,...

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