Nuclear extracts from peripheral blood lymphocytes

[Emmanuel Skoufos]

On Mon, 24 Jun 1996, Chris Hughes wrote:

> Hi there,
> 
> We are having a problem with nuclear extracts.  Using human peripheral
> blood lymphocytes we have tried many of the published "quick/small scale"
> nuclear extract preps with but have yet to find a really satisfactory
> method.  All the detergent methods we have tried (NP-40 based) lead to
> lysis of the nuclei, usually at the point where we add the high salt
> buffer. Consequently (I think) we get low protein yields, usually less
> than 1 ug/ul. Does anyone know of a method that works with PBL to produce
> intact nuclei and high yields (we have no problem with other cell types
> and lines). 
[snip]

I have been using a detergentless protocol described in

Andrews N.C. and Faller D.V. (1991) _Nucleic Acid Research_, *19*:2499

Basically, the cells are osmotically lysed, nuclei are pelleted and 
nuclear proteins extracted with high salt.

I had success in lymphocyte *lines* (Raji, BJAB, PD-31), and in a variety 
of other cell lines and primary cells.  It is brief, can be used with a 
small amount of cells (10^6 -10^7), and its yields are satisfactory for 
my purposes (Gel retardation assays; 100-1000ug per 10^7 cells depending 
on cell type).  It may worth giving it a try :-)


> 
> Thanks
> 
> Chris Hughes
> 
> 

Emmanuel