In article <Pine.3.89.9408301930.A27546-0100000 at carson.u.washington.edu>,
Leon Parker <mrclean at U.WASHINGTON.EDU> wrote:
>for Western blot analysis? I've tried pelleting cells from 1 mL of log
>phase culture, resuspending in 50 uL 2X SDS PAGE loading buffer, freezing
>on dry ice (and I've tried liquid nitrogen), and then boiling for 5-10
>minutes. The yield of total protein using this method is very low
>compared to the extracts I've prepared by smashing the cells with beads.
Try using moe cells in your boiling method. My lab has been doing it
that way for a long time, without much trouble. I often add some PMSF
to the SDS buffer, but I don't think it is important. You can really load
a lot of cells into the buffer, at least up to 1/3 volume. Try useing
10mL of cells at the same OD.
We don't freeze. We just spin down the cells (usually 10 mL), wash
once with water (really just to get the cells into an eppendorf tube),
spin again, and resuspend in 50-400 mL Lemmli buffer. Boiling for 10 or
more minutes takes care of the only problem: DNA coming out of solution
when the gel is loaded.
Unless someone points out a serious problem, this technique is far
more satisfying that other longer techniques.
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