Patricia McChesney/Cellular Fractionation
Cellular Fractionation per Montes de Oca et al. Blood 105(3): 1003. 2005
1. Cells are grown on 15cm dishes, treated with MMC 12hrs, trypsinized and collected by centrifugation.
2. Wash cells once with cold PBS, and aliquot equally into four microcentrifuge tubes.
3. Pellet cells.
- Freeze one pellet in liquid N2
- This is the P1, whole cell pellet
4. Resuspend remaining pellets in cold Buffer A + 0.5% Triton X-100
5. Incubate @ RT 2 minutes to permeabilize cells
6. Centrifuge cells 3 minutes @ 300g, keep the sup, freeze in liquid N2
- This is S2, cytosol and nucleosol
7. Wash pellets with cold buffer A. Freeze one pellet
- This is detergent-insoluble nuclei, P2
8. Digest nuclei with RNase-free DNase 1 in buffer A for 30 minutes @ RT
9. Pellet the residue. Keep sup. Freeze sup. Freeze pellet
- These are S3 and P3, respectively
10. The last pellet is extracted with cold buffer A containing 0.25M ammonium sulfate 5 min @ RT to extract remaining chromatin
11. Centrifuge @ 1877g for 3 min. Keep sup and pellet.
- The resulting pellet is P4, nuclear matrix
- The resulting sup S4 contains chromatin, and the majority of histones
12. Pellet is washed 1X cold buffer A and centrifuged @ 800g 3min.
SDS-PAGE can be performed based on protein concentration, or equivalent cell volumes.