SDS extraction of whole cells

Michael Witty mw132 at mole.bio.cam.ac.uk
Mon Jul 30 08:02:55 EST 2001


Dear Frank,
          is this for bacterial cells or mammalian cells?  Regards Mike.

On Sun, 29 Jul 2001, Frank O. Fackelmayer wrote:

>
>
> 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?
>
> we do this in the following way:
> * harvest cells (no more than 5x10exp6 per sample), spin down in a
> microfuge tube
> * rapidly resuspend cells in 360ul water until you have a homogeneous mixture
> * immediately add 40ul of 10% SDS (i.e. final conc. 1%)
> * sonicate briefly to shear the DNA (resulting solution should not be
> highly viscous any more)
> * add 400ul of Methanol, vortex vigorously
> * spin down for 1 minute
> * add 100ul of Chloroform, vortex vigorously again
> * spin down for 1 minutes
> * (proteins form an interphase between chloroform and methanol)
> * remove supernatant almost completely (containing DNA, lipids, and low
> molecular stuff like salts)
> * add 300ul of Methanol, vortex vigorously
> * spin down precipitated protein for 3 min
> * remove supernatant and allow pellet to air-dry
> * redissolve pellet in SDS sample buffer
> * boiling and/or sonication may be necessary to bring all proteins back
> into solution
> * spin down for 10min
> * use supernatant for the gel
>
>
> This method usually yield samples with superior quality for SDS gels,
> because DNA and lipids are effectively removed. It is much better than
> using sonication alone.
>
>
> Frank
>




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