Purification of GST-fusion proteins E. coli. (pGEX vector)
Mullins lab 4/30/99
IPTG: 0.1 M in ddH20
PMSF: 100 mM in 95% ethanol
EXTRACTION BUFFER: 1X PBS, 1 mM EDTA, 5 mM DTT, 1 mM PMSF
1. ALWAYS start with a fresh transformation. Plate the cells on LB-amp plates and grow overnight at 37 C. (If you're not going to use them right away, plate the cells and keep them at 4 C. Shift them up to 37 C the night before you need them. Put TPM culture medium at 37 C to warm up overnight.)
2. Pick about 200 colonies. Be careful to pick individual colonies and don't just rake the plate.
3. Put colonies into 0.5-1.0 ml of LB in a sterile eppendorf tube. Mix by inversion and innoculate into 1L warmed culture medium (TPM or M9LB).
4. Incubate at 37 C with shaking until cells reach an OD600 of 0.4-0.6.
5. Induce expression with 0.1 mM IPTG (1 ml of 0.1 M stock) for 1-2 hours at 37 C with shaking.
6. Harvest cells by spinning at 5000 RPM for 10 minutes. Keep on ice.
7 Wash cells 1X with 100 mls extraction buffer (without PMSF). Freeze pellets if necessary.
8. Resuspend cells in 100 mls extraction buffer with PMSF and lyse with microfluidizer.
9. Spin lysate at 18.5k RPM for 45 minutes and take sup.
10. Load supernatant onto glutathione column or bind to glut beads in batch (ovenight at 4 C is recommended).
11. Wash column with 10-20 volumes of PBS + 1 mM DTT.
12 Elute with 10 mM reduced glutathione (+1 mM DTT) in PBS. (N.B. Be sure to pH the elution buffer to 8.0 after adding the glutathione!) Collect 1-2 ml fractions and assay by A280 or Bradford.