3) Group 9 in section A03 was frustrated when they did not purify enough of thei
ID: 276056 • Letter: 3
Question
3) Group 9 in section A03 was frustrated when they did not purify enough of their fluorescent protein unknown to get an absorption spectrum. Their bacterial plates contained lots of brightly colored colonies, but the protein seemed to have gotten lost during the purification They run the SDS-PAGE hoping for an explanation, and get the following coomassie stained gel. Monome Position Lane key 1. Crude lysate 2. Unbound lysate 3. Elution 2; heated a. What went wrong during the fluorescent protein purification with the nickel resin (i.e where is the protein)? (You may assume the protein was not degraded by proteases.) b. If all of the reagents for the lab were out on the front table right next to each other, and group 9 was rushing through the purification so that they could leave lab early-a cautionary tale here!-what is one plausible explanation for how the problem occurred.Explanation / Answer
Please find the answers below:
Answer a: According to the information, the protein extracted failed to show positive result on SDS-PAGE right after the initial step of purification i.e. using nickel resin. The nickel resin might have degraded the protein thus resulting in failure of obtaining any protein in SDS PAGE. Secondarily, the protein might have strongly bound to the nickel resin itself and failed to elute out of the resin column. Thus, the eluent as well as the flow-through did not contain any protein.
Answer b: The crude protein sample contains large amount of cellular proteases which are active under room temperature. Since the experimental processes were performed in hurry, the sample might not have been kept on ice throughout and thus, intracellular proteases might have degraded the protein.
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