Nucleosome isolation

How were the nucleosomes isolated? How does the sucrose gradient work?

Nucleosome Isolation Protocol (McNurry and Krangel, Science, 2000)

1. Filter thymocytes

2. Centrifuge at 350G/7min

3. Resuspend cells with RBC lysis buffer for 5min at 23*C

4. Wash twice and resuspend with lysis buffer and equal volume of 0.04% NP-40 for 5 min at 4*C

5. Pellet nuclei through 30% sucrose by centrifuging at 350G/7-14min

6. Wash and resuspend in digestion buffer

7. Incubate with micrococcal nuclease at 1000U/mL for 10 minutes at 37*C

8. Shift to 0*C and add 5mM Na2EDTA to stop digest

9. Centrifuge at 13000G/1m at 4*C

10. Collect supe and add NaCl (50mM)

11. Remove H1 linker histone by incubatating with 30mg/mL Sephadex CM-25 for 1.5h at 4*C

12. Make a linear sucrose gradient 10-45% by layering 40% on bottom, 10% on top

13. Load 2-3mg of sample

14. Centrifugate 40,000rpm/18hours in SW-40 rotor : Components of loaded material will move until they reach the gradient containing the same weight

15. Collect fractions

16. Analyze fractions by subjecting aliquots of each fraction to SDS-PAGE gel

Check for presence of H2A, H2B, H3, H4, and absence of H1

(Schnitzler, Current Protocols in Molecular Biology, 2000)