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?
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.