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E. coli lysis for Protein Isolation- URGENT - CORRECTION

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Tue Jan 18 03:54:39 EST 2000


Michael Jones wrote:

> Hi
>
> Sorry, but I forgot to mention in the original posting that I need the
> lysate to bind to glutathione-agarose beads as well.
>
> below is the corrected version of my request.
>
> I am running a student practical next week, and I no longer have access
> to a sonicator for making cell lysates from E. coli.
>
> The experiment simply involves growing E. coli containing a pGEX fusion
> construct, lysing the cells, and analysing the cleared lysate on an
> SDS/PAGE gel and staining with Coomassie blue.  We also use the lysate
> to run on another SDS/PAGE and western blot and probe with a monoclonal
> antibody against the GST protein.
>
> The lysate is also used to bind to glutathione agarose beads to select
> for the GST-fusion protein, which is also run on the SDS/PAGE gels.
>
> The experiments work very well.
>
> This time round I do not have access toa sonicator to lyse the E. coli
> cells.
>
> I usually work with a 2 ml culture of E. coli.
>
> CAN ANYBODY HELP?
>
> What I need is a simple method for lysing the cells that doesn't use a
> sonicator.
>
> It would be nice if it is simply a chemical disruption with relatively
> simple lab raegents, as opposed to a commercial reagent.
>
> I need the lysate able to be used to bind to glutathione-agarose beads.
>
> The proteins we are looking at are soluble.
>

Hi Michael,
You can try B-Per from Pierce (no affiliation!). It does a good job for
extracting soluble proteins from E.coli. I don´t exactly know what´s in
it, but you can play around with (buffered) sodium deoxycholate, it may
behave similarly.

Frank







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