Nuclear Extraction - (Nov/05/2001 )
Can someone tell me how to do nuclear extraction from mammalian cells? Thank you!
HiI am doing nuclear extraction pretty often and I am using this protocol, which works for me really well. Reagents: Lysis Buffer: 10mM Hepes pH 7.5, 2mM MgCl2, 1mM EDTA, 1mM EGTA, 10mM KCl, 1mM DTT, 10mM NaF, 0.1mM Na3Vo4, 1x Pharmigen-Protease - CocktailNuclear extraction buffer (NEB): 25mM Hepes pH 7.5, 500mM NaCl, 1mM DTT, 10mM NaF, 10% Glycerol, 0.2% NP40 5mM MgCl22ml cell culture spin down (1500 rpm, 5'). Add 400 ul Lysis Buffer, resuspended cells and incubate for 15' on ice gently mix the tube from time to time after 15' add 25ul of 10% NP40 , mix carefully and incubate on ice for 5'Vortex 2x 10 sec (max power) and centrifuge for 30 sec (13000rpm) to pellet the nucleitransfer supernatant (cytosl) to a new tube Add 50ul NEB to the pellet and 2 or 3 time sonicate centrifuge for 5' at 13000rpm, the supernatant contains nuclear proteins
Good luck !!!