Protocol for nuclei extraction or isolation using IGEPAL - (Feb/28/2006 )
Well I guess I could do that, but really that's a lot of work. FACS is just a lot easier (extract nuclei 30 min, run FACS 10min compared to days of work for a southern blot.
I guess I could try adding EDTA, that's supposed to keep cells non-sticky for FACS, so it might work for nuclei as well.
Thanks a lot for your help.
LeserattePD
I see your point that you want quantitative data of nuclei with the oligo and the FACs won't do that. A laser scanning microscope should give you FACS data from slides as it were and I know somebody using it to look at phagocytosis of fluorescent particles combined with a fluorescent membrane stain. If you had access to this type of microscope you could use a fluorescent nuclear stain and look for the amount of oligo present with the nuclear region of staining.
Otherwise making the prep. of nuclei and using a BSA/EDTA buffer in the FACS should work hopefully. There should be a way of distinguishing between clumps of cells and single cells as it's important in analysing DNA content by PI staining.
All the best,
Ceri
HI
Does anyone have a nuclear extraction protocol for neurons? I've tried a protocol similar to the one posted above by aimikins and majority of the cells do not lyse.
Any suggestions
JP2877
jp- is there nothing in the literature? when I came up with that protocol, I found about 7 or 8 papers by others who had done emsas with my cell type; I picked and chose and combined and threw it together based on the properties of all the different components, and it worked. have you tried a lit search yet?
Actually that's how I put my protocol together. I probably just need to lower the osmolarity of Buffer A and I should be able to get the cells to lyse and not the nuclei. I'll do another search to see what I can find
Thanks
JP
I aslo try to seperate cytoplamic and nuleus- protein with this method.....I am using hepg2-cells...but I want to recue the cytoplasmic protein as well.....but when I do this type of extraction some stange things happen....
Nucleus extract seems to be grat in protein conc......but with the cytoplasmic protein I do a TCA- purification step and get a pellet...but when I solve it in buffer ...the protein yeald is amazing low....but I would expect a much higher amount of protein in the cytoplasmatic fraction, right ????
Maybe the lysis conditions are a little bit to soft for hepg2.....??? I realy woul be happy about a good guess.....
Thanx