Pellet cells by centrifugation at 2000 g for 5 min @ 4°C
Resuspend pellet in 1 ml Sample Buffer (1ml for a well of 6-Well plate).
Sonicate twice 10s level 1 (be careful to not generate an emulsion). Stay at 4°C
Add 50 mM DTT (50 ml of 1M DTT in 1ml)
Incubate 10 min @ room temperature on nutator
Add 2 M thiourea (TU; 150 mg to 1 ml)
Incubate 10 min @ room temperature on nutator
Add AG 501-X8-(D) (bio-rad) 5 g/ml (50 mg to 1 ml)
Incubate 1h @ room temperature on nutator
Transfer 200 ml to another tube and add 1X ampholytes (bio-rad)
Rehydrate strips (invitrogen) with 160ml of sample in tray/cassette (invitrogen) for an hour on rocker at room temperature (could be done overnight).
Set up IEF run as recommended by invitrogen. Wet whatman paper placed at electrodes with 700ml deionized water. Use 600ml deionized water as running buffer.
DO NOT USE DISTILLED WATER!

Run (step voltage):
200V for 20min
450V for 15min
750V for 15min
2,000V for 1h to 3h
Prepare SDS-PAGE gel for 2nd dimension electrophoresis.
Wash the strip 10 min in equilibration buffer I
Discard carefully as much as possible (by aspirating) equilibration buffer I and wash the strip 10 min in equilibration buffer II
Rince in running buffer (only few seconds) and mount the strip on SDS-PAGE gel (bio-rad). Orientate the strip from ladder / basic (-) / acidic (+) end.
Seal the strip with 0.5% agarose/bromophenol blue/SDS-gel running buffer
Load the ladder and run the gel
Perfom Western Blot if necessary