SDS extraction of whole cells

Frank O. Fackelmayer Frank at Fackelmayer.de
Sun Jul 29 12:32:25 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?

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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