Microplate gel-filtration method for radioligand-binding assays

A thin-layer gel-filtration chromatographic method has been developed in a 96-well format to separate free and protein-bound ligand in radioligand-binding assays. The mobile phase in the gel-filtration plate is removed via quick centrifugation before samples are applied. Protein-bound ligand is recovered via centrifugation into another 96-well plate for radioactivity measurements. The method exhibits excellent recovery of protein-ligand complexes and less opportunity for dissociation of the complexes since it eliminates major dilution effects from the mobile phase of a column and from elution steps in conventional gel-filtration chromatography. It offers other advantages: simple, rapid, inexpensive, quantitative, and able to handle a large number of samples as required in drug discovery and clinical settings. This microplate gel-filtration method was optimized in studies of receptor-ligand interactions using estrogen receptors as examples and can be used in other radioligand-binding assays.