Answer both parts in details please. You have raised a specific, high-affinity m

Question

Answer both parts in details please.

You have raised a specific, high-affinity monoclonal antibody against an enzyme you are working on for you senior thesis. You further have identified that the antibody binds a stretch of six amino acids of the enzyme. Your advisor suggests that you could use the antibody to purify the enzyme by affinity chromatography from a tissue preparation in which you have lysed the cells. This technique involves attaching the antibody to an inert matrix in a column. You pour the cell lysate over the column, allowing the antibodies to bind your enzyme but not other proteins. The enzyme can then be eluted off the column using a solution of the hexapeptide corresponding to the binding site. The main advantage of affinity chromatography is that it allows rapid, one-step purification under mild conditions that do not denature the protein. In a preliminary experiment you show that if you incubate the antibody with the hexapeptide at room temperature, it no longer can bind the enzyme. So the enzyme and the hexapeptide do, in fact, complete for the antibody?s binding site. Thus encouraged, you bind a fresh batch of the antibody to the column matrix and show that it completely removes your enzyme from the cell lysate. When you try to elute the enzyme off the column with a concentrated solution of the hexapeptide, however, you recover little of the enzyme. What could have gone wrong? How can you make it work? (40 points)

Explanation / Answer

The preliminary experiment shows that the hexapeptide could not bind the antibody at room temperature.

During purification, little enzyme was recovered. For elution, hexapeptide might be added at room temperature (RT). The hexapeptide could not bind the antibody at RT, it could not compete for the antibody’s binding sites. Due to lack of competition, the enzyme could not be displaced.

To make the purification a success, preliminary experiment needs to be done to confirm the optimum temperature at which the hexapeptide binds to the antibody with high affinity.

Suppose, the hexapeptide binds to the antibody at 40 C. Then, for elution of enzyme, the temperature is to be maintained at 40 C to allow the hexapeptide to compete for the binding sites and bind to the antibody.

The binding temperature of hexapeptide could be above of below RT (maximum chances are that the temperature will be above RT). By optimizing the hexapeptides binding temperature, the problem can be solved.

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