Question

1. What are the experimental limitations of this binding assay? List ways to fix them

Answer 1

The aim of binding assays is to measure interactions between two molecules, such as a protein binding another protein, a small molecule or nucleic acid. Hard work is required in preparing reagents but flaws in the design of many binding experiements limit the information obtained.

Consider basic reversible, bimolecular binding reaction:

A+B AB

An ideal binding experiment has a fixed total concentration of one of the two reactants. Lets choose A, that is lower than the K_d (equilibrium dissociation constant). Ten fold lower than the K_d is fine. Using the low concentration of A simplifies the execution of the experiment and the interpretation of data, because essentially all of the other components of B are free in all other conditions.

The practical limitation is that the assay must be able to measure the concentration of free or bound A.

If one chooses a low concentration of total A, then the experiment is to determine the extent of reaction over a wide range of concentrations of B. When the concentration of B is below the K_d, most of the B is free, because, given the low concentrations of both the reactants, little binding will occur. At higher concentrations of B, more AB forms, but the concentration of A is so small that virtually all of B is free. Because most of the B is free both below and above the K_d,

B_freeB_Total one does not have to compensate for any B bound to A when analysing the data.

If the asay lacks the sensitivity to do the experiment with a concentration of A below the K_d, then use the lowest practical concentration of A. later, when analysing the data, compensate for the finite amount of B bound to A at each concentration of total B in the sample.