Berglund Lab: Protease Purification
- For information see: Danielle Cass Lab Book 1/5/04 (Dates 7/15-7/20)
- Vector: Pgex4Tl (BamH1, EcoR1 insert sites)
Transform plasmid into BL21 competent cells (Day 1).
Overnight Culture (Day 2)
- Start a 50mL culture of 2xYT with 100μg/mL Amp (50μL 1000x Amp stock).
- Pick one colony from BL21 transformation plate.
- Shake overnight at 37°C.
Large Scale Culture (Day 3)
- Dilute overnight culture to 1L with 2xYT + 100μg/mL Amp (1mL Amp stock).
- Shake at 37°C until OD600 = 1.2
- Add IPTG to 0.25mM (500μL of 0.5M IPTG per 1L Culture)
- Shake at 37°C for 3 hours.
- Spin down cells at 6000 RPM for 15 min and store in -20°C overnight (-80°C if storing longer).
Cell Lysis (Day 4)
- Resuspend cells in up to 45mL Lysis Buffer with 2mM fresh DTT.
- Sonicate cells in glass beaker, on ice.
- Tune sonicator according to directions.
- Sonicate at power setting 7, 3x30sec.
- Spin down in JA-17 12,000 RPM for 30min.
- Save supernatant, discard pellet.
- Wash 5ml packed GST beads (~7.5m-10mL suspended).
- Spin beads down at 1000 RPM for 3 min.
- Remove supernatant.
- Add 45mL Lysis Buffer, mix.
- Spin down, remove supernatant.
- Repeat 3x from adding Lysis Buffer.
- Add supernatant from cell lysis to washed GST beads.
- Rotate 1 hour at 4°C.
- Wash beads 3x with 45mL Lysis Buffer.
- Elute GST-Protein off of beads.
- Add 10mL Elution Buffer
- Shake at RT l0 min.
- Spin down beads at 1000 RPM, 3 min.
- Remove AND SAVE elution.
- Repeat 3x
- Place GST-eluted Protease into 15kd MWCO dialysis tubing.
- Dialyze in 1L Dialysis Buffer for 4 hours at 4°C, change buffer and dialyze overnight.
Usage of Protease (Day 6)
- Take protein concentration. YOU MUST USE A SPECTROPHOTOMETER for quantitation. The Bradford will give you a 10 fold less number.
- Aliquot out ~0.04 μmol protease (~100μL of 400μM).
- Abs coefficient = 5120 M-1cm-1
- Store at -20°C.
Lysis Buffer (500mL)
- 25mM Tris pH 7.5 (12.5mL of 1M)
- 300mM NaCl (30mL of 5M)
- 1mM EDTA (1mL of 0.5M)
- 2mM DTT (0.5mL of 2M) (add after filtering with a .22μm vacuum filter)
Elution buffer (500mL)
- 50mM Tris pH 8 (15ml of 1M)
- 3.08mg/ml reduced glutathione (add fresh)
Dialysis Buffer (1L)
- 150mM NaCl (30mL of 5M)
- 10mM EDTA (20mL of 0.5M)
- 1mM DTT (0.5mL of 2M)
- 50mM Tris pH 8 (50mL of 1M)
- 20% Glycerol (200mL of 100%)
Suggested Cutting Conditions
- 150mM NaCl
- 50mM Tris-HCl pH 7.0
- 1mM EDTA
- 1mM DTT
At 4°C for 16 hours.