Difference between revisions of "Purification of GST Fusion Proteins"

Revision as of 15:12, 6 May 2009

Bacteria Production and Induction

1. Express and induce protein in culture under appropriate conditions (normally do the following)
2. grow an overnight culture in ~25 mL LB/Amp (+Chloramphenicol if needed) from a colony <2 weeks post transformation
3. add 5 mL culture to 1L TB/Amp and grow at 37C
4. grow to OD600 of 0.6-1.0 and induce with 10 uM IPTG (optimize for each protein)
5. let grow O/N at <25C (optimize induction time/temp for each protein, see Induction Conditions).
6. centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point

Lysis and Purification

1. Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification
2. French Press cells 2 x 15 000 psi (see French Press Protocol)
3. Centrifuge lysate at >15 000 RPM for 30 min to clarify
4. Add ~0.5 mL glutathione-agarose to 50 mL PBS (no PI) to prepare beads. Pellet beads 5 min at 1000 RPM and remove buffer
5. Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
6. Incubate with rotation for 1h at 4C
7. Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
8. Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 5 mL of PBS
9. Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay
10. Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
11. Measure protein concentration (Bradford or A280; concentrate if necessary and store at -20)
12. Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE