Nuclear extracts from peripheral blood lymphocytes
cchughes at uci.edu
Mon Jun 24 16:17:14 EST 1996
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). Would other detergents be better? Or perhaps freeze-thaw? --
douncing is not really practicable with the relatively small numbers of
cells we use (1 E7). Would longer incubations in high salt (say 2 hours
rather than 15-20 min), even with lysed nuclei, produce better results? We
plan to try the latter this week.
Any comments, insights or protocols gratefully received.
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