A fast ultrafiltration method is described for quantitative studying of protein-ligand interactions at equilibrium. This protocol is aimed for student studies in the laboratory.

Bovine serum albumin is allowed to complex with phenol red dye at various concentration ratios in a series of tests. These equilibrium mixtures are then subject to quick ultrafiltration steps using centrifugal ultrafiltration filters. The dye concentrations in the filtrates are determined by spectrophotometry, thus establishing the concentrations of free dye in equilibrium with protein bound dye in each case. In combination with knowledge of the protein concentration and the total amount of dye in each case, it is possible to evaluate the number of bound phenol red molecules per protein molecule.

In a typical experiment a saturation curve, and a Scatchard plot were constructed using results from 10 different equilibrium mixtures. The results typically show that a total of 13-14 phenol red molecules bind to serumalbumin by two interacting sites with different binding constants.