Analytical size exclusion chromatography
Complex formation of Rab proteins preparatively loaded with GppNHp and the bMERB effector domains was assessed by analytical size exclusion chromatography (aSEC). The effector domains were used at a concentration of 113 µM, Rab proteins were used at concentrations of 130 µM (Rab:effector stoichiometry of 1.2:1), 180 µM (1.6:1) or 250 µM (2.2:1) and 30 µl of the protein solutions were injected into a Superdex S75 10/300 GL gel filtration column (flow rate 0.5 ml/min, detection of absorption at 280 nm, buffer: 20 mM Hepes pH 7.5, 50 mM NaCl, 2 mM DTE, 2 mM MgCl2).