Problems with nuclear extraction - (Dec/15/2009 )

Dear fellow scientists,

For a western blot assay on nuclear NFkB, I need the nuclear fraction of PBMC. I used commercial kits of both Cayman and Pierce for the nuclear isolation of 2 x 10^6 cells. My yield is only ~200 ng in total for both cytoplasmic and nuclear fraction for both kits. Do you have any idea how to get a higher yield; so what are the major pitfalls and the most critical steps?

kind regards,

Kornelis

in my experience, absolutely the most important thing you can do is to keep everything very cold. very, very cold.

I chill pipettes and everything for extractions. I work over a big flat tub full of ice; all my tools, buffers, and tubes are pre-chilled in a -20, then stuck into the ice until I use them, on ice. I've found this increases yield quite a bit, in NHEK anyways.