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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



Histone

MW (kDa)

H1

21.5

H2A

14.1

H2B

13.8

H3

15.3

H4

11.3

(Schnitzler, Current Protocols in Molecular Biology, 2000)