tokens: 75 cm2 of 90 % confluent cells were harvested in 500 micro l lysis buffer ( 50 mM HEPES , 150 mM NaCl , 10 % glycerol , 0 . 5 % Triton X - 100 , 2 mM MgCl2 , 5 mM EDTA ) , and the cytosol was incubated for 90 minutes at 4 degrees C with 50 micro l 50 % Glutathione Sepharose beads ( Amersham Biosciences ) linked to anti - V5 antibody ( Invitrogen ) .