Currently, I am performing cell fractionation to separate cytoplasmic and nuclear fractions to quantify the localization of my protein. Right now, I am test the fractionation method.
I am using GAPDH (thermo) and H3 (abcam) as loading control for cytoplasmic and nuclear fractions. GAPDH looks pretty clean, exclusively in cyto fraction. However, H3 has very strong signal in both cyto and nuclear fraction. Anyone has the same problem? Is there any good cell fractionation protocol?
I also tried abcam HDAC1 antibody. It's much better than H3. At least part of the reason is the low abundance of HDAC1 compared to H3. cyto fraction is relatively clean. However, that antibody is relatively expensive. 100 ul for $400, 1:1000 dilution only. Does anyone know a better antibody for HDAC1?
btw, any recommendation for a vendor of H3 antibody?
Thanks,
I appreciate it!
Hello,
I would suggest you to check our paper in Cancer Research (PNUTS acts as proto-oncogene by sequestering PTEN in nucleus )
What fractionation protocol are you using? I use the NE-PER kit from Thermo and have never really had problems with contamination.
Cell type can affect fractionation alot. Percoll gradients generally give cleanest mitochondrial fraction. In my experience it is never perfect and contamination exists. Call it fraction "enrichment " and accept some contamination if it doesn't affect your question. If you absolutely need pure fractions use different approach- confocal localization etc!
you can use lamin B or Lamin A/C. I use Lamin B for nucleus and alpha tubulin for cytoplasm.
http://www.nature.com/cddis/journal/v4/n3/full/cddis201363a.html
You can see methods section from this article. After resuspension, incubate on ice for 5 mins (critical step). Spin at 2000 rpm for 10 mins to get the nucleus. Further spin the sup at high speed to remove debris and get CE. After washing I sonicate and go for a high speed spin of my nuclear pellet in Buffer C to get NE.
I have been using this protocol for 5 years and never face problem. The article is from our lab.
Hi Niels,
I am using online recipes. basically use hypotonic buffer to lyse cells and then high salt buffer plus glycerol to nuclear protein extraction. Thanks for the information on the kit. I suspect it will be more expensive
Hi Gary,
Thanks for the information. I used HepG2 cells. I did notice it's different from 239 cells.
+1 for the NE-PER kit from Thermo, it has always worked very well for a few of us in our lab. As for nuclear control, we've tried Lamin B (Source: CST), B-actin (Source: SC) and Histone H3 (Source: CST). Lamin B is a bit unpredictable and shows inconsistencies in our case ((we treat neurons & astrocytes with H2O2 as a form of injury), B-actin in much more reliable especially in astrocytes while Histone H3 apparently works best for neurons. Point is: the nuclear loading control that'll work for your purpose will need to be figured out from trying a lot of different suggested ones..
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