Cell Fractionation

Material: Cell Fractionation Kit (CST #9038)

1. Prepare cell sample from 75T culture at about 80% confluency.
2. Prepare all buffers fresh immediately before use.
3. For Cytoplasmic Isolation Buffer (CIB), prepare 500uL per sample.
4. Add Protease Inhibitor 1 10uL.
5. Add PMSF 10uL.
6. For Membrane Isolation Buffer (MIB), prepare 150uL per sample.
7. Add Protease Inhibitor 1 10uL.
8. Add PMSF 10uL.

Cytoplasmic fraction isolation

1. Wash the cell pellet with PBS.
2. Resuspend the cell pellet in 300uL CIB and vortex for 10 seconds.
3. Incubate on ice for 30 minutes and vortex for 10 seconds every 10 minutes.
4. Centrifuge at 500 x g for 5 minutes.
5. Carefully collect supernatant as cytoplasmic fraction and avoid disturbing the pellet.
6. Wash the pellet with 100uL CIB.
7. Vortex for 10 seconds.
8. Centrifuge at 500 x g for 5 minutes.
9. Discard supernatant as completely as possible, even if some pellet is lost.