To evaluate PB-FRET as an assay method, we suggest the customer use a known assay from another screening method. In Format 2, the labeled reactants normally bind with one another, while the analyte is an inhibitor of this binding event. In Format 3, the binding of labeled reagents to an intermediary molecule is inhibited by the analyte.
Many variations are possible.
Several strategies are available for labeling the biomolecules in the binding pair, based on the specifics of the assay and the preferences of the investigator. ProZyme supplies RPE and APC Conjugation Kits with the dyes chemically activated for conjugation to immunoglobin molecules or other sulfhydryl-containing proteins. If both reagents are to be indirectly labeled, of course, it is necessary to employ a different pair of secondary reagents for each molecule. Table 1 summarizes the various reagents available for labeling. Once the necessary reagents are in hand, it is simple and straightforward to determine whether FRET-producing interactions take place.