Glutathione sepharose 4b slurry beads

HEK 293 cells were transfected with GST-tagged constructs for 24 h. Cell lysates were harvested. Thirty micrograms of total lysates was kept to determine the expression of the respective constructs (input) and the remaining lysate was incubated with 50 μl of Glutathione Sepharose^® 4B slurry beads (Amersham Biosciences). Beads were washed thrice with mammalian cell lysis buffer. Bound proteins were recovered from beads by adding 50 μl of 2× SDS sample buffer and analysed by western blotting.
For immuno-precipitation assay, HeLa cells were lysed with mammalian cell lysis buffer and 30 µg of total lysates was kept to determine the endogenous protein levels (input). The remaining cell lysates were pre-cleared with irrelevant antibody slurry beads and incubated with antibodies to proteins of interest or control antibodies, which were immobilized on magnetic Protein A slurry beads (Merck Millipore). Beads were washed four times with mammalian cell lysis buffer. Bound proteins were recovered from beads by adding 2× SDS sample buffer. The supernatants recovered were analysed by western blotting.

GST fusion constructs were expressed in BL21 E. coli bacteria, and crude bacterial lysates were prepared by sonication in TEDGN (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 0.4 M NaCl) in the presence of the protease inhibitor mixture. In vitro, transcription and translation experiments were done with rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. Briefly, equal amounts of GST fusion proteins were immobilized on 50 μl of 50% glutathione-Sepharose 4B slurry beads (Amersham Biosciences) in 0.5 ml of GST pull-down binding buffer (10 mM HEPES, pH 7.6, 3 mM MgCl₂, 100 mM KCl, 5 mM EDTA, 5% glycerol, 0.5% CA630). After incubation for 1 h at 4 °C with rotation, beads were washed three times with GST pull-down binding buffer and resuspended in 0.5 ml of GST pull-down binding buffer before adding 10 μl of in vitro transcribed/translated proteins for 2 h at 4 °C with rotation. The beads were then washed three times with the binding buffer. The bound proteins were eluted by boiling in 30 μl of 2× sample loading buffer and resolved on SDS-PAGE.

GST fusion constructs were expressed in BL21 E. coli bacteria, and crude bacterial lysates were prepared by sonication in TEDGN (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 1 mM dithiothreitol, 10% (v/v) glycerol, 0.4 M NaCl) in the presence of the protease inhibitor mixture. In vitro transcription and translation experiments were done with rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. Briefly, equal amounts of GST fusion proteins were immobilized on 50 μl of 50% glutathione-Sepharose 4B slurry beads (Amersham Biosciences) in 0.5 ml of GST pull-down binding buffer (10 mM HEPES, pH 7.6, 3 mM MgCl₂, 100 mM KCl, 5 mM EDTA, 5% glycerol, 0.5% CA630). After incubation for 1 h at 4 °C with rotation, beads were washed three times with GST pull-down binding buffer and resuspended in 0.5 ml of GST pull-down binding buffer before adding 10 μl of in vitro transcribed/translated proteins for 2 h at 4 °C with rotation. The beads were then washed three times with binding buffer. The bound proteins were eluted by boiling in 30 μl of 2 × sample loading buffer and resolved on SDS-PAGE^{61 (link)}.