What protocols are good/best for separating nuclear and cytoplasmic fractions in stem cells or cells with a large nuclei:cytoplasmic ratio?

09 September 2015 3 7K Report

I've been trying to fractionate glioblastoma stem cells (essentially transformed neural stem cells/progenitor cells) using either this Nature Methods protocol: Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading.

the NEB-PER kit: https://www.thermofisher.com/order/catalog/product/78833 supplemented with proteinase and phosphatase inhibitors.

I keep getting very pure nuclear fractions (no cytoplasmic or ER associated proteins) but my cytoplasmic fractions are always positive for Histone H3, but not PARP1 interestingly. I also get spliced GAPDH RNA only in the cytoplasmic fraction while I see 7SK nuclear RNA in both. I have either discovered that these cells secrete mini nuclei (possible but unlikely) or my fractionation is disrupting the nuclear membrane and letting nuclear proteins leak into the cytoplasm (more likely).

-Troubleshooting:

1. Ive tried centrifuging the cells to pellet them at very low speeds. I've used very dilute amounts of NP40 (0.01%), and higher or lower quantities of the CERI than suggested. I still get nuclear contamination.

2. The cells have very large nuclei, cell swelling in the hypotonic or CERI buffer isn't really apparent, the cells are larger but not prodigiously.

3. Ive tried washing the pellet a few times before proceeding to cell swelling. I don't know a good way to remove dead cells which may contaminate my cytoplasmic fraction.

Edward Emmott

While my fractionation was for looking at proteins rather than RNA, I've had a lot of success on a variety of cell lines (though not stem cells) using Angus Lamonds lab protocol - http://www.lamondlab.com/pdf/CellFractionation.pdf

Western blotting using Tubulin, GAPDH (cytoplasmic) or lamin B1, HNRNP proteins gave clean results.

Christine Klaus

Years ago, I used the Dignam Prep to isolate individual nuclei, and cytoplasmic fraction. It is very old school, but great fun to see the nuclei isolated by phase contrast. Maybe this simple protocol will be robust enough for your needs.