Maleimide-mediated conjugation of Alexa-488 to EC1236C/44W

Alexa-488 dye (Thermo Fisher Scientific) was stably conjugated to the thiol groups of cysteine of the purified EC12_36C/44W with a maleimide reagent (Life Technologies) according to the manufacturer’s instructions. This reaction was carried out at 4°C with mixing of Alexa-488 and EC12_36C/44W peptide at an equimolar ratio overnight in buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl and 1 mM tris (2-carboxyethyl) phosphine. Both dithiothreitol and β-mercaptoethanol should not be used in the reaction because each of them contains free thiols. Labeled protein was separated from free dye molecules by size exclusion chromatography (Superdex 200). The fluorescently labeled EC12_36C/44W (EC12 probe or probe) was aliquoted and frozen in −80°C for in-solution and competitive binding assays.