Purification of GST Fusion Proteins

Revision as of 15:07, 5 May 2009 by Davebridges (Talk | contribs) (Created page with '==Bacteria Production and Induction== #Express and induce protein in culture under appropriate conditions (normally do the following) #grow an overnight culture in ~25 mL LB/Amp ...')

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Revision as of 15:07, 5 May 2009 by Davebridges (Talk | contribs) (Created page with '==Bacteria Production and Induction== #Express and induce protein in culture under appropriate conditions (normally do the following) #grow an overnight culture in ~25 mL LB/Amp ...')

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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