Site-specific Protein Labeling for FRET

AF488 maleimide or AF647 maleimide (Jena Bioscience) was added in a tenfold molar excess to Archease-S147C (50 µM) or RTCB (50 µM), respectively. RTCB was labeled in the presence of Mn²⁺ (500 µM) and GMPCPP (500 µM). After 2 h incubation at room temperature, the reaction was stopped with an excess of β-mercaptoethanol. The sample was applied onto a desalting column (HiTrap Desalting, Cytiva) and eluted with desalting buffer (25 mM HEPES, pH 7.3, 250 mM NaCl₂, 5% Glycerol, 1 mM TCEP) to remove excess dye. Labeled Archease-S147C or RTCB was flash-frozen in liquid nitrogen and stored at 80 °C, protected from light.