Pad 11:31 PM * 8%) 1) In the lab you are characterizing a helicase from E. coli.
ID: 220081 • Letter: P
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Pad 11:31 PM * 8%) 1) In the lab you are characterizing a helicase from E. coli. The active protein is a hexamer composed of identical 50kDa subunits. The gene encoding this protein was inserted into a pET vector so that it could be expressed with a N-terminal 6x His-tag. Following Ni- chromatography you analyze a sample of the helicase by SDS PAGE and then use the gel in a Western Blot with a primary monoclonal antibody (conjugated to an appropriate infra-red fluorophore) that recognizes the 6x His-tag. a) If the helicase is 100% pure AND intact (i.e. not degraded) after Ni-chromatography, how many bands and of what size do you expect to detect on the Western blot? b) If the helicase is only 60% pure AND intact (i.e. not degraded) after Ni-chromatography how many bands and of what size do you expect to detect on the Western blot? Explain c) You run the sample in (b) on another identical gel but this time you stain it with Coomassie Blue. Would you see the same number of bands as in (b)? Explain d) After Ni-chromatography the helicase is 100% pure but about 30% of the total protein in your sample has been degraded at 4 different sites at the C-terminal end. How many bands do you expect to detect on the Western blot? Explain 2) In a separate experiment you overexpress Enzyme X. At the end of the protein prep your sample only contains the enzyme i.e. it is 100% pure and does not contain any protein contaminants. In addition it has the expected 100% activity and has not been degraded Your protein also does not contain any post-translational modifications e.g. phosphorylation. You analyze two identical aliquots of your enzyme on a Coomassie Blue stained SDS and Native gel. Your results are shown below 150kD 120kDa 80kDa 40kDa On both gels lanes X and Y contain identical samples of pure protein 20kDa SDS-PAGE Native Gel Using the data above determine the structure/composition of the enzyme. In your answer identify each band on each ofthe gels and explain why these bands are present and how you have used them to determine the composition of the enzyme.Explanation / Answer
Answer 1 a) In SDS-PAGE proteins are denatured and separated according to their respective sizes. So if helicase proteins is 100 % pure and intact after SDS-PAGE it will show only 1 band of 50 KDa in western blot due to denaturation of hexamer protein.
b) If the expressed helicase proteins is 60 % pure and intact still after SDS-PAGE it will show only 1 band of 50 KDa in western blot because the antibody used against his-tag will specifically recognize only hos tag and thus helicase giving one band of 50 KDa in western blot.
c) If the sample (b) is run into SDS-PAGE and then stained with coomassie blue then this dye will stain all the proteins present in that gel. Since the sample is only 60% pure and rest other proteins of different sizes are there, the gel will show multiple bands (protein contaminant) along with the expressed helicase.
d) If the 100% pure but 30% degraded helicase sample is run into SDS-PAGE and detected via western blotting using antibody against his-tag present at the N-terminal, then it will show 5 proteins bands out of them 1 would be of complete non-degraded protein (70%) and 4 will be result of degradation at 4 sites at C-terminal end.
Answer 2) lets analyze first SDS-PAGE, we got 2 bands of different molecular weight of approximately 80kDa and approximately 50kDa in SDS-PAGE. This indicates that the active enzyme is dimer of two subunits (A and B for assumption). Now if we see the bands in native gel there are 3 bands present. Since there is no denaturation so each of the three band in native gel corresponds to one dimer. Now since we got 3 bands which indicates that there are three different dimer. This could be possible only when both subunits are interacting with themselves (A/A or B/B) and with other subunits (A/B). This suggest that the enzyme is heterodimer.
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