Patent ID: 8859295

Claim:
A method of determining the dissociation constant (K d ) for a particular protein and ligand combination, comprising: providing a first solution comprising said protein with no ligand and a predetermined minimum amount of chemical denaturant; and a second solution comprising said protein, no ligand and a predetermined maximum amount of said chemical denaturant; mixing said first and second solution in a plurality of samples, each having said first and second solution in different proportions to create solutions having different molarities of said chemical denaturant; measuring an observable property of each of said plurality of samples; creating a denaturation graph based on said observable property and said molarity; determining the molarity at which a predetermined fraction of said protein molecules became denatured (F d ); preparing a third solution comprising said protein and a ligand at a specified concentration ([L]) and an amount of chemical denaturant equal to said determined molarity; measuring said observable property of said third solution to determine a fraction of said protein molecules that became denatured in the presence of said ligand (F d,l ); and using said specified concentration ([L]), said predetermined fraction (F d ) and said fraction of said protein molecules that became denatured in the presence of said ligand (F d,l ) to determine said dissociation constant for said particular protein and ligand combination, wherein said dissociation constant (K d ) is calculated from the formula: K d = [ L ] A * 1 - F d , 1 F d , 1 - 1 , where A is defined as F d 1 - F d .