I am trying nucleolar isolation from hela cells.
Do you have any specific questions? Do you have kits available to you? Otherwise, a good, generalized protocol can be found here:
http://www.lifetechnologies.com/us/en/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction-method-.html
I've had OK success with the protocol and it circumvents the need to buy kits, though those admittedly may give better results.
From the page (buffer recipes at that page):
1. Collect cells (5 x 106) in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent).
2. Wash cells twice with cold PBS.
3. Remove and discard the supernatant and collect the cell pellet.
4. Transfer the cells into a prechilled microcentrifuge tube.
5. Gently resuspend cells in 500 μl 1x Hypotonic Buffer by pipetting up and down several times. Incubate on ice for 15 minutes.
6. Add 25 μl detergent (10% NP40) and vortex for 10 seconds at highest setting.
7. Centrifuge the homogenate for 10 minutes at 3,000 rpm at 4°C.
8. Transfer and save the supernatant. This supernatant contains the cytoplasmic fraction. The pellet is the nuclear fraction.
9. Resuspend nuclear pellet in 50 μl complete Cell Extraction Buffer for 30 minutes on ice with vortexing at 10 minute internals.
10. Centrifuge for 30 minutes at 14,000 x g at 4°C. Transfer supernatant (nuclear fraction) to a clean microcentrifuge tube.
11. Aliquot and store at –80°C. The nuclear extracts are ready for assay.
You may see Nuclei Isolation Kit : Nuclei EZ Prep available from Sigma-Aldrich for details.
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