Subcellular fractionation

1. Grow 1-2 15cm plates of 293T/HeLa cells per condition
2. Aspirate media from plate. Stand plate on side for 10 seconds to remove final traces of media.
3. Add 5mL of PBS per plate. Use cell scraper to gather cells. Add to 50mL Falcon tube. Recover remaining cells with 5mL PBS.
4. Centrifuge at 4°C for 5 min at 500x g.
5. Aspirate supernatant. Add 5mL PBS to wash cells and repeat spin.
6. Aspirate supernatant. Transfer cells to 2mL Eppendorf tube. Repeat spin.
7. Estimate packed cell volume (PCV)
   1. I add water to another 2 mL tube to estimate the PCV.
8. Add 3x PCV Buffer A to wash out PBS. Carefully resuspend cells in Buffer A with wide bore pipet tip. Take some cell suspension as input!
9. Spin at 4°C for 5 min at 500x g in microfuge. (The cells will have begun to swell by this time.)
10. Aspirate supernatant. Add 5x PCV of Buffer A (+ protease inhibitors). Incubate on ice for 10min to swell.
11. Add 10% Triton X-100 to final concentration of 1% 
    Rock sample in hand for 1 minute.
12. Attach 25G needle to plunger. Wash needle 1x with Buffer A.
13. Slowly pull suspension through needle 6-7 times, trying not to generate bubbles
    - Sometimes after 1-2 strokes, cellular material will not enter needle. Proceed anyway.
14. Spin sample at 4°C for 5 min at 1500x g. The supernatant is the cytoplasmic extract. Wash the nuclei 1x in 500uL Buffer A. Repeat spin.
    1. I try to resuspend the nuclei with wide-bore P1000 pipet tips.
15. Remove supernatant. Estimate pelleted nuclei volume (PNV)
16. Resuspend nuclei in ½ PNV of low salt Buffer B. 
    - Use a wide bore pipet tip to resuspend the nuclei as best as possible. 
    - Be careful not to over-handle the nuclei, so that they don’t lyse.
17. Once nuclei are resuspended, add high salt Buffer C drop-wise to the suspension.
    -Typically, add 4 “drops” of buffer to suspension, close cap and carefully invert tube to mix. Repeat procedure until all Buffer C is added.
    -Don’t add protease inhibitors to high salt Buffer C so that it’s salt concentration is not altered.
18. Nutate nuclei at 4°C for 30min
19. Spin at full-speed for 20min. Supernatant is high salt nuclear extract.
20. Wash pellet 1x with Buffer A (likely won’t resuspend very well). 5min spin at full speed. Aspirate supernatant.
21. Resuspend pellet in 600uL of RIPA buffer.
22. Sonicate sample with probe sonicator
    5x, 17% amplitude, 20s ON, 40s OFF
23. Spin full speed 10min 4C. Supernatant contains nuclear pellet material.

Buffer A: 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, protease inhibitors

Buffer B (low salt): 20 mM Tris-HCl pH 8.0, 25% glycerol, 1.5 mM MgCl₂, 20 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, protease inhibitors

Buffer C (high salt): 20 mM Tris-HCl pH 8.0, 25% glycerol, 1.5 mM MgCl₂, 1.2 M KCl, 0.2 mM EDTA, 0.5 mM DTT

RIPA buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% SDS