Nuclear extraction on non-primary culture cells - Western blot (Dec/08/2010 )

Hi

Would anyone know how to extract nuclear proteins from neural stem cells and/or peripheral blood mononuclear cells?

I've been having trouble getting a signal for this protein of interest, and I've tried troubleshooting everything from harvesting whole-cell lysate to western blotting (e.g. increase protein concentration loaded into each well, increase antibody concentration, change buffer concentrations, wash more/less, etc).

Or if someone can recommend a good company, that'd be a great starting point too. I'm actually looking into Millipore's protocol, but still wanted more suggestions/advice from the general crowd.

Thanks!

I would think that a straight forward cellular fractionation protocol would work with these cells. Try the one published by Mendez and Stillman, it's quite easy and fast. Although this protocol is intended to look specifically at chromatin-bound proteins, you would be more interested in the P1 pellet (see Figure2 in the attached paper) and you could just lyse the nuclei in RIPA and spin out the DNA.

rkay447 on Wed Dec 8 15:41:45 2010 said: