Reaction-Diffusion Based Measurement of Protein-Ligand Affinity

Many methods to measure protein ligand binding/dissociation constants require an intrinsic shift in a “signal” from the protein (i.e. fluorescence, NMR spectra, etc.), thus they require some optimization for each protein measured. However, by using reaction-diffusion (RD), one can accurately measure protein-ligand dissociation constants, Kd, for arbitrary protein-ligand combinations.

In this talk, we show that Kd may be measured using a general RD process. Here, we first “ink” a patterned agarose stamp with a protein, then apply the stamp to a poly(acryl amide) gel which contains immobilized ligand. As the protein diffuses from the stamp into the gel, it intereacts with the ligand: when the protein is bound to the ligand it is stationary, however, when it is not bound to the ligand it freely diffuses. After a prescribed period of time, the stamp is removed from the gel, and the gel is stained with either Coomassie blue or a combined Coomassie blue/silver staining method. The resulting pattern of stained protein may then be fit using the RD equations to determine the dissociation constant for that protein/ligand pair.