You have raised a specific, high-affinity monoclonal antibody against an enzyme

Question

You have raised a specific, high-affinity monoclonal antibody against an enzyme you are working on, and have identified its interaction site as a stretch of six amino acids in the enzyme. Your boss suggests that you could use this antibody to purify the enzyme via affinity chromatography. In this technique, the antibody is attached to an inert matrix of a column, a crude cell lysate is passed over the column, allowing the antibody to bind the enzyme but other proteins, and finally eluting your enzyme by washing the column with a solution containing the six amino acid peptide corresponding to the binding site. In preliminary studies, you show that if you incubate the antibody with the peptide concentration corresponding to the binding site it will no longer bind your enzyme. Encouraged, you bind the antibody to the column and show that it completely removes your enzyme from the crude cell lysate. However, when you try to elute you enzyme with a solution containing a high concentration of the peptide, none of the enzyme comes off the column. What could have gone wrong?

Explanation / Answer

A situation in which no enzyme is eluted by altering the composition of elution buffer may have occured in a situation when either the conditions of elution buffer (containing the peptide) like pH are not suitable for proper interaction of the stationary phase or the elution buffer.

What may have also happened is that, due to high concentration of the peptide taken in the elution buffer, the intermolecular interactions of the peptide has increased (peptides are closer to each other due to a concentrated solution) as a result of which the peptide sequence is no longer free to interact with the antibody and help in elution of our enzyme.

This however is all a part of troubleshooting and needs to be validated using different experimental setups.

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