Liver sauce (2 litres)

320mM sucrose (219g)

25mM Tris base (6.05g), pH 7.4 with HCl

Breaking buffer (2 liters):

500mM KCl (74.55g)

250mM sucrose (171.15g)

25mM Tris base (6.05g) pH 8.0 with HCl

2mM EGTA (1.52g)

0.5mM 1,10-phenanthroline (stock at pH 5)

1mM DTT*

1mM PMSF (0.5M stock in DMSO)*

* add immediately before use

Dialysis buffer (4 litre volumes)

50mM KCl (14.9g)

25mM Tris base(12.11g) pH 8.0 with HCl

1mM DTT

450g of tissue- store tissue cold in sauce imediately after slaughter

All steps at 4°C

Add 450g to 1200ml capacity Waring blender, fill with breaking buffer, add PMSF. 2 x 30 second bursts.

Repeat with second 500g.

Pool homogenates.

Centrifuge at 9000g for 1 hr And recover supernatant (decant through cheesecloth).

Dialyse using Spectra/Por 2 (6.4ml/cm) twice for 2hr. min. vs 30l of dialysis buffer.

Change dialysis buffer, dialyse 4-12 hour versus 30l.

Spin at 120,000g for 1hour at 4°C (45K 50.25 rotor).

Collect supernatant. Store at -80°C

OR

Add ammonium sulfate to 60% saturation slowly over 20-30 minutes. Add 0.345g/ml.

Recover precipitate by centrifugation 25,000g 15 minutes.

Resuspend in dialysis buffer to 1/7 original dialysis volume.

Dialyse vs. 3x4litres dialysis buffer.