You are working in the lab at night trying to make progress on your cell-free sy
ID: 254214 • Letter: Y
Question
You are working in the lab at night trying to make progress on your cell-free system. Since your immense brain requires extra ATP, you go out very briefly for a donut, leaving:a mixture on ice containing membrane + cytoplasm from VSV-infected PaBo cells that have been pulse labeled and washed to remove the 35S methionine. You return after a few minutes and begin your immunoprecipitations over the usual 16-minute time course. In the middle of your experiment your labmate approaches you and admits to being a true pabo. While you were eating your donut, he had accidentally added membranes from uninfected WT HeLa cells to your mixture: Membrane fraction from uninfected WT HeLa cells (ndo pulse label) Membrane cytoplasm from Vsv-infected PaBo cells after 35 S Met pulse labeling Mixed :( After expressing your frustration, you decide to finish the experiment anyway and observe the following results Unprocessed Processed VSV-G 10 12 14 16 35 Time post''S Met washout(min) A. Please provide two plausible explanations for the results. Be as detailed as you can given the information available to you. 10 ptsExplanation / Answer
In case of top bands, as indicated its unprocessed VSV-G sample, top band of western blot represented the free heavy chain molecule of the antibodies, which are higher molecular weight protein molecule that were abundant (unbound stage) at the initial. Later as the incubation proceeded, the immunoprecipitation was significant due to antibody binding to the target protein and precipitation was effective resulting in the increased amount of the immunoprecipitation for the target protein. Therefore, the bandwidth of target protein presented in the western blot analysis was increased across time of precipitation as the time interval increases.
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