TEV preparation - Séraphin Lab
j-1
Transform BL21/LysS strain on KanR with optimized TEV plasmid (stock pBS2440).
j
Inoculate 25 ml medium A
1)
with one colony at the end of the afternoon.
Grow at 37°C.
j+1
Inoculate 1 L medium A with the 25 ml preculture.
Grow at 30°C until OD600 reach 0.9.
Add IPTG to the medium up to 0.1 mM.
Grow 6 hours at 23 °C.
Pellet cells 20 min at 4,000 rpm in GS3.
Wash with PBS 1X.
Freeze pellet in liquid N2 and store at -80°C.
j+2
Resuspend pellet in Buffer 1
2)
Adjust to 20 ml with Buffer 1.
Pass through French-Press 2 times.
Spin down with ss34 12,000 rpm for 30 min.
Put the supernatant with 1 ml slurry of NiNTA beads (previously washed with buffer 1) in a Falcon tube.
Rotate 1h at 4°C.
Transfer in an Econo-column (Biorad).
Wash with 15ml Buffer 1.
Wash with 5 ml Buffer 2
3)
wash with 10 ml Buffer 1.
Elute with 4×0.5 ml Buffer 3
4)
in 0.5 ml glycerol tube (50% glycerol final).
store at -80°C.
You may perform an additional chromatography step fort higher purity.
1)
LB, Cm 25 μg/ml, Kan 10 μg/ml
2)
20 mM Tris-HCl pH 8.0, 10 mM imidazole, 200 mM Nacl, 2 mM β-ME, 0.2% NP40
3)
20 mM Tris-HCl pH 8.0, 10 mM imidazole, 1 M NaCl, 2 mM β-ME, 0.2% NP40
4)
20 mM Tris-HCl pH 8.0, 200 mM imidazole, 200 mM NaCl, 2 mM β-ME, 0.2% NP40