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1) Suppose you have identified a 20-nt DNA sequence that, from genetic experiments, has been implicated as a transcriptional enhancer of a gene of interest. You want to identify the DNA-binding protein(s) in a crude HeLa nuclear extract that binds to it. You perform an EMSA with a highly radioactive form of the 20-mer double stranded DNA and see a band shift. Which of the following methods could you use to help identify the protein or proteins causing the gel shift? Explain why the method could or could not be used and if it can what are its limitations.
a) mass spectroscopy
In this method, 20-nt DNA sequence can be incubated with the crude HeLa nuclear extract. In this case, the radioactive label is not appropriate, that is the reason why this method is not the best one in this example. The DNA sequence should be biotinylated and pre-conjugated to streptavidin-coated beads. Washing step could be applied to eliminate nonspecifically bound proteins. Specifically bound proteins could be eluted with SDS-page sample buffer with boiling. The protein can be identified by excising the electrophoretic band, extracting the protein, trypsin hydrolyzation, and application of mass spectroscopy for peptide identification. The limitation of this method (except the different labeling of the oligonucleotide) is the sensitivity. EMSA can detect amounts of protein on the order of picograms, but mass spectroscopy usually needs protein in nanogram amounts. Also, sometimes working with nuclear extracts, non-specific DNA binding proteins must be removed by passing lysates over other DNA sequences that do not bind the same protein....
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