sentence: Purified GST-Ubc13 ( 1 µ g ) was bound to glutathione-Sepharose , washed and preincubated for 1 h with varying concentrations of compounds Ia IIa , or a control cyclic compound , and then allowed to bind Uev1 ( 1 µ g ) for 1 h in a buffer containing 50 mM Tris-HCl pH 7.6 , 0.1 mM EDTA , 150 mM NaCl and 0.2 mM DTT .