Membrane binding assays for peripheral proteins

Membrane affinity of peripheral proteins can be measured by several different methods. The fluorometric assay, either intrinsic tryptophan intensity measurement or FRET, is the simplest method and should thus be the first one tried. In the case of low fluorescence signal, alternative methods need to be selected, depending on the membrane affinity of protein and the availability of instrument. For enzymes that can be readily quantified by activity assays, either direct methods or separation assays can be used. The centrifugation method with lipid-coated beads (or vesicles) would serve as a rapid and inexpensive assay for these proteins. For nonenzymatic peripheral proteins, the centrifugation assays are not recommended because of low sensitivity of protein determination. The SPR analysis has emerged as a universal assay for all peripheral proteins, but careful experimental controls and data analysis are required to obtain meaningful parameters from the assay. The calorimetric method can also serve as an accurate assay for peripheral proteins with modest affinity. The lipid monolayer technique is a useful assay to determine the ability of a protein to penetrate into the membrane. Finally, it is recommended that for a given protein Kd be determined by an assay be validated by a second assay performed under the same experimental conditions.