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purification:tev:s_protocol

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TEV preparation - Séraphin Lab

j-1

  • Transform BL21/LysS strain on KanR with optimized TEV plasmid (stock pBS2440).

j

  • Inoculate 25 ml medium A1) 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 12)
  • 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 23)
  • wash with 10 ml Buffer 1.
  • Elute with 4×0.5 ml Buffer 34) 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
purification/tev/s_protocol.1468424418.txt.gz · Last modified: (external edit)