Nuclear Protein Extraction

1. Wash cells twice with 10ml ice cold PBS

2. Scrape cells into 5ml ice cold PBS and rince plate with 5 ml

3. Transfer cells into 15ml tube, spin 1000 rpm, 5 min at 4C

4. Resuspend pellets in 1ml ice cold PBS and transfer to 1.5 ml tube, spin down for 10s in microfuge (20 000 g)

5. Resuspend pellets in 400 ul ice cold buffer A

6. Swell on ice for 15 min, vortex 10s

7. Pellet nuclei for 10s in microfuge (20 000 g)

8. Resuspend nuclei pellets in 25-40 ul of buffer B

9. Incubate on ice for 20 min, flicking tube occasionally

10. Pellet debris for 5 min at 4C (20 000 g)

11. Save and aliquot supernatant, store at -80C

Buffer A: 10mM HEPES, pH 7.9; 1.5mM MgCl2; 10mM KCl; 0.5mM DTT; 0.2mM PMSF + proteinase inhibitors +phosphatase inhibitors

Buffer B: 20mM HEPES, pH 7.9; 25% glycerol; 420 mM NaCl; 1.5 mM MgCl2; 0.2 mM EDTA; 0.5mM DTT; 0.2mM PMSF + proteinase inhibitors +phosphatase inhibitors




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