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

As this is an example of competitive inhibitors. So using high concentrated solution of the hexapeptide may decrease the elution. In this method, the concentration of competitive agents should be added equally to the concentration of the coupled ligand.

However, if the free competing compound binds more weakly than the ligand to the target protein, use a higher concentration of competitive agent to achieve efficiency in elution.

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