Problems with nuclear extraction (View forum version)



Kornelis

Posted 15 December 2009 - 01:59 AM

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

aimikins

Posted 15 December 2009 - 04:23 PM

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.