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