Tween-40 Membrane Prep

All steps on ice

  • Wash cells and adjust to 10^8 to 10^9/ml in 140mM Tris/Saline
  • Make up protease inhibitors:
  • PMSF, 0.0174g/ml of DMSO, isopropanol or acetone
  • IAA, 0.09625g/ml water
  • Aprotonin (bought as solution)
  • used at 5µl/ml
  • Add an equal volume of 5.0% Tween-40 in 140mM Tris/Saline containing 2 x protease inhibitors ie 10µl/ml each.
  • Incubate at 4°C for 1 hr with mixing.
  • Homogenise in hand held homogeniser for 6 strokes (may check by viewing a drop under a microscope for intact cells)
  • Spin out nuclei at 1500g, 5 minutes
  • Retain S/N, resuspend pellet in