The picture depicts a NATIVE PAGE taken after each step of a protein purificatio

Question

The picture depicts a NATIVE PAGE taken after each step of a protein purification experiment. Here is the description of the protocol followed: You started with salt fractionation, followed by affinity chromatography. You then purified the sample using anion-exchange chromatography, collecting early and late fractions. You measured the activity of your sample after each step and found a low specific activity after the salting out step, and high specific activity after affinity chromatography, but no activity in any of the collected fractions after the anion-exchange step. However, combining early + late fractions from the anion exchange purification results in a very high specific activity. What is the best explanation for these observations? Explain

Explanation / Answer

The low specific activity after the salting out step signifies that the protein is less soluble in high salt concentrations as the additional charges of the salt concentration shield the protein resulting in aggregation and precipitation. The affinity chromatography shows high specific activity, this signifies that the protein sample being purified does contain protein molecules that are required for the protein interactions such as transcription factors. The anion-exchange chromatography signifies that the protein sample contains the proteins with a net negative charge. The binding affinities of the different proteins affect the strength by which the protein binds to the resin and thus facilitating the separation at different phases. When the sample is separated, the early and late fractions looses their catalytic property to a certain extent during the purification, but when the fractions are mixed it is possible that the fraction complements their activity and suggests that the protein sample being separated contains protein interaction molecules and negatively charged protein molecules which show interaction when combined and when separated shows no activity.

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