Brain sauce (2 litres)

320mM sucrose

25mM Tris-HCl, pH 7.4

Breaking buffer (2 liters):

500mM KCl

250mM sucrose

25mM Tris-HCl (pH 8.0)

2mM EGTA

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

1mM DTT*

1mM PMSF (stock in ethanol)*

* add immediately before use

Dialysis buffer (30 litre volumes)

50mM KCl

25mM Tris-HCl (pH 8.0)

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 9000g for 1hour at 4°C.