Glutathione coated beads

Exponentially growing cultures E. coli DH5α cells containing either the pVOS33 or pVOS34 plasmid were treated with 1 mM isopropyl-β-d-1-thiglucopyranoside (Roche Applied Science, Basel, Switzerland) for 2 h at 30°C. Cells were then harvested by centrifugation at 5500 rpm for 15 min at 4°C and lysed by sonication in lysis buffer (50 mM phosphate buffer, pH 8.0, 300 mM NaCl, and 10 mM imidazole). After centrifugation at 10,000 rpm for 10 min at 4°C, supernatants were collected and incubated in batch with nickel-nitriloacetic acid agarose beads (Qiagen, Limburg, The Netherlands) at 4°C under continuous rotation. After 2 h, beads were collected by centrifugation and washed three times in lysis buffer with 20 mM imidazole, and proteins were eluted in lysis buffer containing 250 mM imidazole. The GST-Rac1 binding domain of Pak1 protein was induced as described and purified using glutathione-coated beads (GE Healthcare). Beads with the purified protein were stored frozen until experimental use. In all cases, protein concentration was determined by running aliquots of both the purified His-tagged protein and GST fusion protein–coated beads in SDS–PAGE in the presence of known concentrations of BSA as standard and stored at –20°C until further use.