Difference between revisions of "Purification of GST Fusion Proteins"

Revision as of 15:07, 5 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).
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