SDS extraction of whole cells

Karl Fischer fischer_karl at hotmail.com
Wed Aug 1 16:39:30 EST 2001



Warren Gallin wrote:

> Hi folks,
>         I am trying to do a Western blot on a set of cell lines and am trying
> to keep the protein concentration as high as possible.  The problem is
> that the DNA released on boiling in SDS makes the sample impossible to
> pipette accurately.
>         Aside from dilution, does anyone have a way to get the viscosity of
> this kind of extract down to a workable level?

If your proteins of interest are not nuclear then you may wish to try the
following:

Collect your cell pellet and resuspend in lysis buffer (10mM Tris-HCl [pH 7.5],
50 mM NaCl, 1 mM EDTA,  8% sucrose, 0.25% NP-40).  Mix by inversion for 3-5
minutes at room temperature then clear the insoluble material by centrifugation
(5-10 minutes).

I usually use 1ml of lysis buffer per 60mm dish if I am lysing cell monolayers
in situ. For cell pellets a 0.5-1 ml volume of buffer is typically adequate for
roughly 10^7 cells. Your mileage may vary ;-)

Regards

karl
U of Alberta
fischer_karl at hotmail.com







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