Pre equilibrated glutathione sepharose 4b resin

Protein extracts from cells transfected with the different versions of GFP-CCAR2 were prepared in lysis buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 0.5% Triton, 50 mM NaCl, 1 × protease inhibitors (Roche) and 1 × phosphatase inhibitor cocktail 1 (Sigma)). The amount of expression of each CCAR2 fragment was calculated by western blotting. Similar amounts of each CCAR2 truncated version were used for pull-down assays. After adding beta-mercaptoethanol (final concentration 10 mM) to the samples, cell extracts were pre-cleared by incubating with 50 μl of pre-equilibrated glutathione sepharose 4B resin (GE Healthcare) for 1 h at 4 °C.
Eighty pmol of purified GST-CtIP were resuspended in a final volume of 500 μl with PBS, mixed with 100 μl of pre-equilibrated glutathione sepharose 4B resin and incubated for 1 h at 4 °C. The resin was washed twice with binding buffer (lysis buffer with 10 mM beta-mercaptoethanol but without protease and phosphatase inhibitors) and then incubated with the pre-cleared cell extracts for 2 h at 4 °C. The matrix was washed twice with wash buffer (binding buffer with 3 mM reduced glutathione), and proteins were eluted by boiling the slurry for 5 min in protein-loading buffer. Precipitated proteins were separated by SDS–PAGE, transferred to PVDF membranes and analysed by western blot analysis.

Eighty pmol of purified GST alone, GST-CtIP or GST fused to CtIP fragments were resuspended in a final volume of 300 μl with PBS, mixed with 100 μl of pre-equilibrated glutathione sepharose 4b resin (GE Healthcare) and incubated for 1 h at 4 °C. The resin was washed twice with binding buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 10 mM beta-mercaptoethanol, 0.5% Triton and 50 mM NaCl). One hundred pmol of purified His₆-CCAR2 was incubated at 4 °C for 1 h with either GST or GST-tagged proteins bound to resin. The matrix was washed twice with wash buffer (binding buffer with 3 mM reduced glutathione). Bound proteins were separated by SDS–PAGE (7.5%), transferred to polyvinylidene difluoride (PVDF) membranes and analysed by western blot analysis using antibodies against CCAR2.

GST, GST-EVH1, GST-Dm-EVH1^L70A, GST-Smk-1, GST-Hs-EVH1 and GST-Hs-EVH1^L69A were expressed in E. coli SixPack strain [52 (link)] and purified to homogeneity as follows. Cells were grown in 50 ml LB medium to A₆₀₀ approximately 0.6 and expression was induced with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside for 5 h at 25°C. Cells were lysed by ultrasound disruption in 30 ml phosphate buffered saline (PBS) supplemented with 1 mM PMSF and 0.2 mg ml⁻¹ lysozyme, followed by centrifugation at 16 000×g at 4°C for 15 min. The cleared supernatant was loaded onto pre-equilibrated glutathione sepharose 4B resin (GE Healthcare) and incubated at 4°C for 1.5 h. Then, beads were washed five times with PBS supplemented with 0.05% Triton X-100, immobilized proteins were kept on beads and stored at −20°C in PBS supplemented with 50% glycerol.