Glutathione sepharose 4b bead slurry

Immunoprecipitation assay was performed using MCF7 cells transfected with plasmids. Whole cell lysates were incubated overnight with 20 μl of protein A/G PLUS agarose (Santa Cruz) or glutathione Sepharose 4B bead slurry (GE Healthcare), at 4°C. Bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot analysis using appropriate antibodies (Santa Cruz Biotechnology).

Human SAMHD1 was cloned into pGEX-6P-1 with an N-terminal GST tag (GE Healthcare, provided by Dr. Yoshio Koyanagi) and transformed into BL21 (DE3) pLysS competent cells (Invitrogen). Cells were grown at 37°C to an A600 of 0.5, stored on ice for 2 h, and induced overnight with 0.25 mM isopropyl-α-D-1-thiogalactopyranoside at 25°C. Cells were harvested and lysed in lysis buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 2 mM EDTA, 1 mg/ml chicken egg white lysozyme, and one tablet of Roche Applied Science Complete protease inhibitor mixture) for 4 h on ice. Cell debris was removed by centrifugation at 49,000 × g for 15 min, and lysate was incubated overnight at 4°C with 1.5 mL of Glutathione Sepharose^® 4B bead slurry (GE Healthcare). Beads were pelleted and washed three times with wash buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 1 mM dithiothreitol, 0.5% Triton X-100), equilibrated in buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20% glycerol, 0.5% Triton X-100) and packed into a column. The column was washed three times with 30 mL of equilibration buffer, and SAMHD1 was eluted with 50 mM Tris-HCl [pH 8], 1 mM EDTA, 10% glycerol, 300 mM NaCl, 200 mM reduced glutathione. A Millipore Centricon protein concentrator (45 MWCO) was used to concentrate the protein and for buffer exchanges. Protein samples were snap frozen using liquid nitrogen and stored at -80°C until use.