Quantitative determination of protein stability and ligand binding by pulse proteolysis

Pulse proteolysis exploits the difference in proteolytic susceptibility between folded and unfolded proteins for facile but quantitative determination of protein stability. The method requires only common biochemistry and molecular biology lab equipment. Pulse proteolysis also can be used to determine the affinity of a ligand to its protein target by monitoring the change in protein stability upon ligand binding. The Basic Protocol describes the detailed procedure for determining protein stability using pulse proteolysis. For pulse proteolysis to be used for determining a protein's stability, the protein should not be digested significantly by pulse proteolysis when it is in the folded conformation. The Support Protocol describes a procedure for determining whether a protein satisfies this requirement. The principles of protein stability determination using denaturant and pulse proteolysis are also discussed.