Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins

Abstract

We studied the activity of the fluorescently labeled membrane transporter MalGFK2, which transports maltose at the expense of ATP hydrolysis. We used a commercially available malachite green assay (SensoLyte MG phosphate assay kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis. However, strong variations in phosphate concentration were measured when using the supplier’s handling protocol. We optimized the protocol, taking into account the effects mediated by glycerol, SDS, and fluorescent label on the sample. As a result we obtained highly reproducible phosphate concentration values under conditions optimal for solubilized membrane proteins.