Hey everyone,
I've had some troubles with my protocol of nuclear fractionation and this forum was recommended. The protocol is based on hypotonic cell lysis with RBS followed by Nonidet P-40 treatment and recovery of nuclei by centrifugation. However, after that I can't suspend nuclei pellet in glycerol-containing buffer, even after pippeting and vortexing. Using a T-75 flask (~4 million cells), this protocol yielded ~20ug of nuclear proteins. Is it normal? Why is nuclei pellet insoluble? Should I sonicate it or use a motor pestle? Help me to solve my problem.
Thanks.
Hello Jonas, what type of nuclear fractionation would you like to do? I do have a protocol in which you will get the separated: cytoplasmic fraction, the nuclear soluble fraction and finally the chromatin-associated fraction. Via this protocol the nuclear soluble fraction is soluble in glycerol-containing buffer whereas the chromatin-associated fraction will be insoluble. Therefore, my question would be what are looking for? To me 20 µg nuclear protein out of 4 million cells sounds reasonable and in a similar range as I am receiving.
Hi Nicole, thanks for comment! I wanna just to fractionate nuclear and cytoplasmic proteins to evaluate MAPK p38 translocation to nucleus. In fact, my trouble resides in the insolubility of nuclei pellet in glycerol-containing buffer. What do you think about using a pestle to smash insoluble chromatin globs? Can solubilize these globs in an adicional buffer?
You will not get the chromatin fraction smashed by a pestle. Why do you want to solve it in an glycerol-containing buffer? What are your purposes after getting it solved? If you just want to run a western blot you can solve the pellet directly in 1x SDS-sample buffer (laemli buffer).
Actually, this protocol is for preparation of nuclear proteins for EMSA (that's not my objective), so it's important to make all chromatin associated fraction soluble. As I need to separate nuclear and cytoplasmic proteins only for western blot, I'll test this week to solve the nuclei pellet directly in 1X LB, as you suggested.
That sounds good, that you do need it just for western blot. You need to "resuspend" the pellet in ddH2O. At this point it will not get solved, then add the same amount of 2x LB, mix it a bit and directly boil it at 95 degree for 5-10 min, check if it is getting solved and otherwise add a bit more 1x LB and then boil it again. Good luck!
Hey Nicole, I performed the nuclear fractionation as you suggested, in comparison to standard one, but it didn't work well. Even I saw ~5 times more protein (ponceau) when compared to "glycerol-extracted" nuclei pellet, western signal for phosphorylated ATF2 was weaker in nuclei "resuspended" in LB 1x! I haven't understood it, because they were the same sample divided in two just before proceeding. I also observed cytoplasmic contamination (beta-tubulin) only in LB-resuspended nuclei. Anyway, thanks a lot for helping.
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