Malachite green phosphate assay

GTPase activity of purified guanylate binding proteins were performed by estimating inorganic phosphate liberated from GTP during enzymatic reaction. In brief, 0.5 μM of each protein/protein combinations were incubated with 150 μM of GTP in buffer containing 20 mM HEPES [pH 7.4], 500 mM NaCl, 5 mM KCl and 10 mM MgCl₂ for 60 min at room temperature. Reactions were quenched by the addition of EDTA. Amount of inorganic phosphate was estimated by a malachite green assay kit according to manufacturer’s instructions (Sigma, MAK307).