Sub-cellular fractionation

Virginia Urquidi vurquidi at worf.molbiol.ox.ac.uk
Tue Apr 1 10:21:31 EST 1997


In Article <whc23-0104971105490001 at powercenter-604.physiol.cam.ac.uk>,
whc23 at cus.cam.ac.uk (Dr.W.H. Colledge) wrote:
>Does anyone have a simple, foolproof (preferably in a single Eppendorf
>tube) method for obtaining a cytoplasmic and nuclear extract from tissue
>culture cells.

I use the following protocol:

Washed pellet of cells.
Resuspend in TNE- 1% NP-40 (or 0.5% NP-40)
(TNE = 10mM Tris-Cl (pH 8.00)/100 mM NaCl/1 mM EDTA)
Leave on ice for 5 min with occasional agitation.
Nuclei are removed from the lysate by centrifugation 
(1500 xg for 15 min 4 C)

Works always well. The nuclei pellet is compact and very white.
If it is not, probably the lysis was incomplete.

Best luck.
Virginia Urquidi 







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