2.5.1.60 | fresh plant material is frozen in liquid nitrogen, ground on ice with acid-washed sand in 100 mM Tris buffer, pH 7.8, containing 5 mM EGTA, 5 mM EDTA, 10 mM beta-mercaptoethanol, and 10% glycerol, lysate is centrifuged, dialyzed against 50 mM Tris buffer, pH 7.8, containing 10% glycerol, and 1 mM DTT |