120 L fermentor: Cell disruption and purification
 Cell disruption


  • Switch on chiller (4°C)
  • switch on disruptor
  • run 1-2 fillings of water through at 10-20 KPSI (don't go beyond 40KPSI with the pressure adjustment, please)
  • run your sample (foaming)
  • run water through until machine is clean
  • run 20% ethanol through to prevent growth
  • switch off chiller and instrument
  • please be aware that unless disassembled, this machine can not be used for Western blot samples (contamination possible)
  • little pieces of plastic or tissue can block the jet. Easy to fix by taking off the chilled steel dome.
 Example purification (Ni Sepharose FF)
  • 120L BAI cells gave 1 Kg of wet cell pellet, frozen in LN2
  • smash frozen pellet into small pieces, be careful not to get pieces of plastic into suspension
  • add 50 mM Tris pH 8 up to 2L, 20 EDTA-free PI tablets (Roche)
  • stir until completely smooth
  • run through cell disruptor at 20 KPSI, let foam settle for 10 min
  • fill 12 type 19 rotor bottles and balance (because of foam)
  • run two rotors at 19.000 rpm for 1 hour at 4°C
  • decant supernatant and collect
  • run supernatant at 20 ml/min over 50 ml Amersham Ni Sepharose FF
  • follow your usual elution protocol, everything at 20 ml/min.
JL 3/6/2005