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 activity increases as we get a more purified protein. On first step of salting out, the sample was slightly purified therefore, the activity was low. On further purification by affinity chromatography the specific activity increased. However, after anion exchange chromatograhy the proteins get separated based on their net charge. This step may result in separation of co-factors from active proteins or separation of active proteins in the early fractions, which results in no activity at the end. But if the early and late fractions are combined it will give very high specific activity as the combination contais both co-factors as well as purified protien.

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