Good nuclear extract protocol?

Ulf S. Andersson u_andersson2 at yahoo.com
Wed Mar 1 11:50:32 EST 2000


Hi!
This is not the best protocol around but at least it works pretty well for
EMSA and it is quick and easy and that appears to be what you were after. It
is a slight variant of NAR vol 19, pg 2499 (1991)

Micro Prep of DNA binding proteins
Keep samples on ice as much as possible! Or work in cold room

1)  Rinse cells with cold PBS.  Scrape off 500K-10M cells in 1 mL cold PBS.
2)  Pellet cells for 10 s in the microfuge.
3)  Resuspend cells in 400 microL cold Buffer A.  Original protocol
recommended flicking the tube, but this does not work well.  Pipetting
gently seems to be fine.
4)  Incubate on ice for 10 min to allow swelling.
5)  Vortex for 10s.  Spin down in the microfuge for 10s.  Discard the
supernatant.
6)  Resuspend the pellet in 20-100 microL (depending on starting # of cells)
cold Buffer C.
7)  Incubate on ice for 20 min.
8)  Spin in the microfuge (at 4C) for 2 min.  Transfer the supernatant
(which contains the DNA binding proteins) to a fresh tube.  Store at -70C.
Yield is 50-75 microg protein per million cells.

Buffer A:  10 mM HEPES-KOH pH 7.9, 1.5mM MgCl2, 10 mM KCl, 1mM DTT, 1mM PMSF

Buffer C:  20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 1 mM DTT, 1 mM PMSF

Good luck!


Gary McLean wrote:

> Hi
> I'm looking for a quick method to prepare nuclear extracts from cultured
> cells where the protein is then suitable for EMSA. ANy information would
> be appreciated.
> Regards
>
> Gary
>
> --
> Lets go Otago!!





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