2. (25 points) You have raised a specific, high-affinity monoclonal antibody aga

Question

2. (25 points) 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 usinga 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, compete 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 the purification method work?

Explanation / Answer

Affinity chromatography depends on the specific and reversible binding of the antibody to the column matrix, which has the enzyme. The antibody was eluted using the hexapeptide that competes with the enzyme for the antibody. The enzyme linked to the inert matrix in affinity chromatography experiment completely bound the antibody as no antibody was seen cell lysate.

However, it could not be released from the enzyme when eluted with the hexapeptide. So, despite the hexapeptide having the ability to compete with the enzyme for the antibody, the conditions in elution buffer did not favor such binding of antibody to the hexapeptide. The hexapeptide should have been able to displace the enzyme from the antibody. It is possible that the affinity between the antibody and the enzyme is much higher than the affinity of the hexapeptide to the antibody. This is not the scenario as the hexapeptide could displace the antibody from the enzyme completely at room temperature. It is also possible that the antibody aggregated on the column. The conditions of the elution buffer are not optimal for the binding of the antibody to hexapeptide.

The elution can be improved by improving the buffer conditions in the elution buffer. Elution can also be performed at a higher temperature. Alternative elution buffers may be tried or a combination of elution buffers can be used for the elution. pH and temperature will affect the binding of the antibody to hexapeptide as well as dissociation of the enzyme from the antibody.

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