Soybean lecithin

RyR2 was purified using SBP-FKBP12.6 affinity chromatography^{30 (link)} with some modifications. The microsomes were solubilized with 2% (w/v) CHAPS (Dojindo) and 1% (w/v) soybean lecithin (Avanti Polar Lipids) in 1 M NaCl, 20 mM MOPS (pH 7.4), 2 mM dithiothreitol, and the protease inhibitor cocktail for 30 min on ice. After centrifugation at 100,000 × g for 30 min at 4 °C, the supernatant was diluted with four volumes of 20 mM MOPS (pH 7.4), 2 mM dithiothreitol, and the protease inhibitor cocktail, after which it was passed through a 0.45-μm filter and loaded onto a pre-equilibrated 5-mL StrepTrap HP column (GE Healthcare, Chicago, IL) with the bound SBP-FKBP12.6 fusion protein. The column was successively washed with 10 column volumes of (1) wash buffer (20 mM MOPS pH7.4, 2 mM dithiothreitol, and 0.3 M sucrose) containing 0.2 M NaCl and 0.25% (w/v) CHAPS and (2) wash buffer containing 0.5 M NaCl and 0.015% (w/v) Tween-20 (Sigma-Aldrich). The SBP-FKBP12.6-RyR2 complex was eluted with the wash buffer supplemented with 2.5 mM D-desthiobiotin (Iba Lifesciences). After checking the purity by SDS-PAGE, the eluate was quickly frozen in liquid nitrogen and stored at −80 °C until further use.