Determination of Transmembrane Protein Affinities for Solutes by Frontal Chromatography

This chapter exemplifies the use of immobilized biomembrane affinity systems and outlines the advantages of the approach, which may be applicable both to membrane proteins present in natural sources and those expressed in heterologous systems. It describes preparation of stationary phases by use of hydrophobic interaction, entrapment, biotin–avidin binding and adsorption. The chapter presents nonlinear regression analysis of chromatographic interaction data for single- and dual-site binding models. Quantitative biomembrane affinity chromatography is preferably done in the frontal mode since this allows the determination of the amount of operative binding sites and gives the exact elution volumes. Immobilized biomembranes have other application areas than quantitative analysis of biospecific interactions. By biomembrane affinity chromatography, affinities can be determined for one or more membrane protein(s) in a cell, in vesicles of the cell membrane or in proteoliposomes by one and the same method on the same gel bed.