Dual Fluorophore Labeling of KirBac1.1

To randomly incorporate both donor and acceptor fluorophores into the channel, substoichiometric labeling with a mixture corresponding to protein/donor/acceptor ratios of 10⁴:8:4 was performed. A reaction mixture of 200 μL KirBac1.1, 1 μL Cy3B maleimide (GE Healthcare), and 0.5 μL Alexa Fluor 647 maleimide (Invitrogen, Carlsbad, CA) was made and incubated in the dark at room temperature for 1 h. It was then added to Amintra CoHIS resin (equilibrated with 20 volumes of 20 mM HEPES pH 7.5, 150 mM KCl) and incubated at 4°C for 1 h. Affinity purification was performed by washing with 20 volumes of wash buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM DM, 10 mM imidazole) followed by elution with 10 volumes of elution buffer (20 mM HEPES pH 7.5, 150 mM KCl, 5 mM DM, 500 mM imidazole) after a 15 min incubation. The eluate was run through an equilibrated NAP-5 column (GE Healthcare) and six 0.5 mL fractions were collected. Wild-type KirBac1.1 contains no endogenous cysteines, and consistent with previous reports (26 (link)), no background labeling was observed. The labeling efficiency of reporter cysteine mutants was estimated by fluorescent visualization of proteins on an SDS-PAGE gel (Nu-PAGE 4-12% Bis-Tris; Novex, Waltham, MA) and visualized using a Pharos FXTM molecular imager (BioRad, Hercules, CA) with Discovery Series Quantity One v4.6.9 software to determine labeled fractions.