Ultrafiltration Method of Studying Binding of Ligands to Macromolecules

The principles and procedures for studying binding of low molecular weight ligands to macromolecules using ultrafiltration are reviewed. Ultrafiltration methods are much faster and theoretically equivalent to equilibrium dialysis, which is the most commonly used subtractive method. An entire binding isotherm and the effect of free species concentration on the binding equilibrium can be determined with a single run of a few hours duration. The calculations and data analysis, which are normally quite complicated, can be easily performed using popular spread-sheet programs such as Lotus 1-2-3® on a personal computer. The experimental protocol, the control experiments to account for nonspecific binding and the calculation procedures are given in detail using a case study of a typical ligand-protein interaction.