Put on orbital shaker, 90rpm, 37°C.

Split 1:1 approximately every day (but if cells are not growing fast enough-leave longer).

Don't continue with these more than 5 days. try not to have more than 300ml per flask finally as cells will settle at higher volumes.

Day before experiment make up buffers required

for 500ml,

10mM MES-KOH pH 6.6 (0.98g) , 140mM sucrose (23.94g), 2mM EGTA, 1mM MgCl2,(use stock solutions) 75mM KOAc (3.68g).

Add VB buffer salts for 50ml, add 40ml D2O, adjust pH with KOH, make up to 50ml.

Add salts for 50ml , add 40ml D2O adjust pH.

Add 10g Ficoll, stir to disssolve (takes a while) then make up to 50ml with water.

Before starting pour the first gradient 11ml (x2 per 5ml PMS). Add inhibitor cocktail and DTT to gradient buffers.

Take cells approximately 1 litre (60 cells counted using haemocytometer).

Spin down and transfer to 50ml centrifuge tube.

Wash with vesicle buffer (VB; 10mM MES-KOH pH 6.6, 140mM sucrose, 2mM EGTA, 1mM MgCl2, 75mM KOAc).

Resuspend in 5ml of VB supplemented with 1mM DTT and protease inhibitor cocktail.

Homogenise in cold room with cell cracker (smallest ball) until >80% of cells are stained with trypan blue. Approximately 15 passes should be fine. If the

device gets very stiff, take it apart and rinse it, before proceeeding.

Spin at 1500rpm, 4°C (Jouan centrifuge, G08).

Incubate supernatant with 50µg/ml ribonuclease A 30 minutes at 4°C.

Spin at 7000g, 45 minutes (8,500r.p.m. TFT55.38, using inserts), to prepare a post-mitochondrial supernatant (PMS) .

Make a mark indicating the top of the gradient on the centrifuge tube. Add ~2ml of PMS to each gradient. Top up with vesicle buffer.

Spin at 45,000g for 35 minutes (TST 41.14, 19,000rpm).

Aspirate load. Collect top 5ml of gradient, using a pipette.

Dilute x5 with VB++.

Spin at 100,000g (33,000rpm TFT 55.38) for 50 min., 4°C.

Pour second gradient during this spin. 11ml total volume 2%Ficoll/9%D2O-20% Ficoll /90% D2O containing DTT and protease inhibitors. Make a mark at the top of the gradient.

Resuspend pellet with several passages through a 26 gauge needle in 1.5 ml VB++.

Apply to second gradient.

Centrifuge to equilibrium at 76,000g (23,500r.p.m.) for 10-14 hours.

Aspirate load.

Collect 1 ml fractions- can store at this point.

75% TCA stock (50g of TCA plus 39ml water).

Dilute TCA stock (75%) 3 parts TCA 2 parts water.

Add 0.5ml to 1ml fractions and leave in cold room for 60 minutes.

Centrifuge at 18000rpm for 30 minutes using the Kontron centrifuge in Bobs Lab.

Take off supernatant, leaving about 50µl.

Add 1ml cold 15% TCA.

Spin 15 minutes top speed.

Take off supernatant.

Repeat this step for high Ficoll fractions 6-10.

Add freezing cold acetone.

Spin 5 minutes.

Pour off acetone onto a dry tissue.

Repeat this step.

Dry for 10 minutes at 60°C on heating block.

Hydrate with water, then add electrophoresis sample buffer (+DTT).

Add 1µl quantities of 1M unbuffered Tris until sample turns blue.

Shake on vortex for 3 hours.

Heat to 96°C for 10 minutes.

Spin sample prior to electrophoresis and try to avoid getting particulate matter in the gel loading tip.

Coomassie stain as usual prior to silver staining-apparently this produces more uniform silver staining.

For subsequent steps only use millipore water.

Incubate with 1% gluteraldehyde for 30 minutes.

Incubate 3x5 mins with millipore water.

Soak in 5µg/ml DTT for 30 minutes.

Pour off-don't rinse.

Soak in 0.1% silver nitrate for 60 minutes.

Rinse rapidly with water.

Develop in 3% sodium carbonate + 50µl of 37% formaldehyde (per 100ml).

Stop reaction by adding 5ml of 2.3M citric acid in distilled water.

Rinse with water.

Store in 20% ethanol at 4°C.