Difference between revisions of "Purification of GST Fusion Proteins by GSTrap"

Revision as of 17:39, 24 July 2012

Bacteria Production and Induction

• Express and induce protein in culture under appropriate conditions:

1. grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.
2. add 5 mL overnight culture to 1L TB/Amp and grow at 37C
3. grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein)
4. let grow O/N at <25C (optimize induction time/temp for each protein, see Induction Conditions).
5. centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.

Lysis and Preparation

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 (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
4. Prepare, filter and chill 1L of PBS and ~50 mL of 10 mM Glutathione (pH 8.0) in PBS.

FPLC Run

1. Fill trey with ice, or put PBS and Glutathione in ice bucket.
2. Start the pump, by going to
3. Rinse the leads with water and place in PBS (A) and Glutathione (B)

Post-FPLC

1. Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
2. Measure protein concentration (Bradford Assay or Quantification by Absorbance at 280nm; concentrate if necessary and store at -20)
3. 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 purified protein (0.5-10 ug) by SDS-PAGE