Alexa 680 c2 maleimide

Full-length human CPR was expressed, purified, and stored as previously reported.¹⁶ After purification, CPR was labeled with Alexa 680 C₂ Maleimide (Thermo Fisher Scientific, Waltham, MA). Briefly, Alexa 680 C₂ Maleimide was dissolved in 100% HPLC grade acetonitrile, stored at −20 °C in the dark, and used in the following 24 h. CPR was diluted to 15 μM in 100 mM HEPES (pH 7.4) buffer containing 150 mM NaCl and 20% glycerol (v/v). The solution was transferred to a sealed glass vial and gently degassed for 5 min under Ar while kept on ice. The dye was added to the degassed protein containing solution in a 1:1.2 protein:dye concentration ratio and allowed to react under gentle stirring at 4 °C overnight while protecting the reaction mixture from light. The reaction was stopped by addition of a 1 M solution of DTT to a final concentration of 3 mM. The labeled CPR was then purified from the excess dye by using a desalting Micro Bio-Spin column packed with Bio-Gel P-6 (BioRad, Hercules, CA). The gel in the column was suspended in Tris buffer (pH 7.4) but was exchanged with 100 mM HEPES (pH 7.4) containing 140 mM NaCl and 5 mM CaCl₂ (referred to as ER buffer). The degree of labeling was calculated according to the protocol and estimated to be 1. The labeled CPR was then aliquoted, flash frozen in liquid N₂, and stored at −80 °C.