This is a simple protocol for purifying GST fusion proteins expressed using pGEX vectors in E. coli. The final product will be the fusion protein bound to glutathione-sepharose beads (appropriate for GST pull-down assays, etc.). The proteins can also be eluted from the beads using 10 mM glutathione in Tris buffer.
1. Grow overnight culture of DH5alpha cells containing pGEX construct in 3 ml LB + 100 ug/ml ampicillin at 37 C.
2. Dilute 1 ml of each culture to 100 ml LB + ampicillin pre-warmed to 37 C, and incubate (shaking) until OD ~ 0.6 (~ 2-3 h).
3. Add IPTG to 1mM, incubate 4 h.
4. Transfer cultures to centrifuge bottles, centrifuge 10 min, 6000 xg at 4 C.
5. Discard supernatant, store pellet at -80 C if necessary.
Pellets can be stored long-term at -80 C with little effect on yield.
6. Resuspend pellet in 10 ml of ice-cold lysis buffer.
7. Add 1 ml triton buffer to resuspended cells, mix by inversion, keep on ice.
8. Sonicate (power level #3) 3 x 30 sec, placing on ice 1 minute between sonications.
9. Transfer lysate to centrifuge tubes, centrifuge at 10,000 rpm, 10 min, at 4 C.
10. Transfer cleared lysate to 15 ml falcon tubes and add 100 ul glutathione-sepharose beads (200 ul of a 50% slurry), rotate 1 hr at 4 C.
Glutathione-sepharose beads should be rinsed once with lysis buffer before adding to lysate.
11. Wash beads 4x with 1 ml wash buffer (at 4 C).
After each wash, beads can be pelletted by centrifuging for 1 min at 2000 rpm. The total washing time should be at least 20 min.
12. Wash once with 1 ml bead storage buffer, centrifuge and discard supernatant.
13. Add 400 ul bead storage buffer, store at –20 C.
Buffer Recipes
Lysis buffer: (need 10 ml per 100 ml culture )
- 20 mM HEPES-KOH pH 8.0
- 500 mM NaCl
- 0.1 mM EDTA
- 200 ul 50x complete protease inhibitors per 10 ml (add fresh)
Triton buffer: (need 1 ml per culture)
- 20 mM HEPES-KOH pH 8.0
- 500 mM NaCl
- 0.1 mM EDTA
- 10% Triton X100
Wash buffer: (need 4 ml per 100 ml culture)
- 20 mM HEPES-KOH pH 8.0
- 500 mM NaCl
- 0.1 mM EDTA
- 0.1% Triton X-100
Bead storage buffer:
- 20 mM Tris/HCl, pH 7.4
- 50% (v/v) glycerol