Ligand binding protein preferences

A plotting protocol (also known as the method of continuous variation) that provides useful information about protein-ligand and protein-protein interactions. Mole fractions Xa and Xb of two interacting substances, say A and B, are varied such that the total molarity remains constant. Note that Xa = [A]/([A] + [B]) and Xb = [B]/([A] + [B]), such that (Xa + Xb) = 1. If an enzyme prefers to bind AB as a one-to-one complex, then the enzymatic activity will be maximal at a mole fraction Xa of 0.5 (Le., the point at which A and B are present in a one-to-one stoichiometry). Similarly, if AB2 is the active species, then the enzyme will be most active at a Xa value of 0.33. In this manner, the stoichiometry of binding can be readily determined, and the technique can yield information concerning the affinity of the enzyme for the active species. 

Chemically labeling a protein always carries the possibility of disrupting its ligand-binding activity or blocking the epitope of the protein, thereby preventing secondary antibody attachment. Therefore, an alternative method to detect the bonnd proteins in their native form is sometimes preferred. 

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