Nuclear extracts from peripheral blood lymphocytes

[Chris Hughes]

In article <k.b.andersson-0107960921430001 at biokjmac6.uio.no>,
k.b.andersson at biokjemi.uio.no (Kristin B. Andersson) wrote:


> In our hands, the trick was to reduce the NP40 concentration to get intact
> nuclei. Most of the "quick/small scale" protocols worked fine with reduced
> NP40. With the human Reh cell line for example, I used 0.05% NP40 instead
> of the usual 0.5% in the lysis buffer. you can test each cell line
> individually by staining the nuclei with trypan blue to see that the NP40
> concentration is sufficient to permeabilize the plasma membrane.
> 
> Kristin


We will certainly try this but the crucial point here is that we are using
normal peripheral blood lymphocytes which seem to be a lot more
troublesome than cell lines.  Have you used this protocol succesfully with
normal PBL?

Also, we tried a detergentless protocol, as suggested by Emmanuel Scoufos
in this group, but didn't seem to get back much protein that bound to our
probe (AP-1).  It seems that a fine titration of detergent may be in
order.

thanks,

Chris Hughes

-- 
Dr. Chris C.W. Hughes                   
Dept. Mol. Biol. & Biochem.
Biol. Sci. II
Univ. California, Irvine                 cchughes at uci.edu  
Irvine CA 92717                          ph (714)  824 8771