Counterflow rejection of adsorbing proteins for characterization of biomolecular interactions by temperature gradient focusing

A new technique is described for the analysis of small molecules in samples containing serum proteins and for the measurement of the binding of small molecules to serum proteins. The new technique is based on temperature gradient focusing (TGF) and takes advantage of the counterflow used with TGF to exclude serum proteins from the analysis channel while small molecules are focused for detection. The technique is demonstrated for the measurement of the binding constant between a small molecule and serum albumin using both a direct measurement of the free fraction of the small molecule as well as using a competitive binding assay.