Anti gst

Plasmids containing GST-, HIS-, and MBP-labelled proteins were transformed into Escherichia coli strain BL21. Transformants were grown to a concentration of OD₆₀₀ = 0.6 in a 37°C concentrator, and placed in a 22°C concentrator for at least 30 min. Expression of the fusion protein was then induced by adding isopropyl β-D-1-thiogalactopyranoside to 0.5 mM, and shaking for 5–6 h in the 22°C table concentrator. E. coli cells were lysed in Tris-HCl buffer containing 25 mM Tris·HCl (pH 7.5), 150 mM NaCl, and 1 mM DTT. The sample liquid was then sonicated to destroy the cell (cycles of 3 s on, 3 s off for a total time of 2 min). If the resulting bacterial liquid was not clear, sonication was repeated. Equal amounts of the two protein solutions were mixed and centrifuged, and the supernatant was incubated with glutathione agarose beads (GE Healthcare) overnight at 4°C. After spinning (500 g, 5 min, 4°C), the beads were collected and washed five times with buffer containing 25 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1 mM DTT, 1% Triton X100, and 0.1% SDS. Finally, the proteins bound to the beads were separated from the beads by boiling in 2×SDS loading buffer in a 95–100°C water bath. The proteins were separated in SDS-PAGE gels and detected by western blot analysis using anti-GST (Medical Biological Laboratories,1:2000) and anti-MBP antibodies (New England Biolabs, 1:2000).

The following antibodies were used: mouse anti-FLAG/M2, Sigma-Aldrich, F1804; rabbit anti-FLAG, Medical & Biological Laboratories, PM020; goat anti-FLAG, Novus Biologicals, NB600-344; Anti-GST, Medical & Biological Laboratories, PM013; anti-Hsc70, Santa Cruz Biotechnology, sc-7298; anti-NPT2, Abcam, ab33595; anti-Rad1, Santa Cruz Biotechnology, sc-14314/N-18; anti-Rad17, Sigma-Aldrich, R8654; and anti-Rad17-S645, Bethyl Laboratories, A300-153A.