Suppose you are given the assignment of isolating an enzyme, \"enslave\" which i

Question

Suppose you are given the assignment of isolating an enzyme, "enslave" which is secreted in to the fermentation broth by the bacterium 8. techy. the enzyme has an approximate molecular weight of 60,000. You grow 3L of 8. techy which you then spin down in a centrifuge. You discard the broth and weight the cells. You break open the cells with a grinding method and centrifuge the resultant paste. You discard the cellular debris and assay for enslave. You find no activity but your assay works well with store bought enslave. What mistake did you make? Your protein of interest has a PI of 6.3 you run your Ion exchange column Initially at a pH of 4.3, A. what type of beads should you use to bind your protein (Anion/Cation/Neutral)? And in this case, would it be DEAE Sephardi or CM Sephardi beads? B. Give an example of how you can elute your protein off the column

Explanation / Answer

3. Centrifuging the cell debris after the grinding method is wrong step in this assay. As the enzyme in question has a molecular weight of 60,000 which is comparable to the cell debris, so there is possibility that the enzyme was also centrifuged out of the resultant paste.

4. A) Since the PI is 6.3 the protein of interest is acidic in nature so we must use an Anionic binding surface. We should user CM sephadex as it is a cation exchanger while DEAE sephadex is an anionic exchanger.

B) Increase the ionic strength of the buffer by increasing the salt gradient that would elute the protein from the column.

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