An equal volume of buffer 3 was added in aliquots (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF, 1% NP40, protease inhibitor cocktail (Sigma)) and the solution was incubated while shaking for further 30 minutes on ice.