cytoplasmic and nuclear extraction - problem with contamination! - (Aug/10/2011 )
I've been trying to separate the cytosolic and nuclear fractions of my 293 cells. My nuclear fractions are very clean but my cytosolic fractions always seem to be heavily contaminated with nuclear proteins. I have tried the Thermo-scientific NE-PER kit, and is now trying the detergent buffer A-buffer B method. The steps i took to get the cytoplasmic fraction as below:
1. Scrape cells in PBS and pellet by centrifugation.
2. Resuspend and incubate cells in buffer A containing 0.05% NP-40 alternative for 5 min
3. Centrifuge at 2500 x g, 30 s
4. Remove supernatant and transfer to a new tube
5. Centrifuge the supernatant again at 13,000 x g for 30 s, and transfer to another fresh tube.
One thing i also noticed is that even when no NP-40 was added, PCNA can still be found in
the cytosolic fraction, although at a lower concentration. Appreciate any help or advice!!!
You may need to do some sort of density gradient purification to get the cytosolic fraction clean.
I had the same problem with NE-PER on tissue. I couldnt get it right so i abandon the kit and made my own solutions. PM me if you want more details