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E. coli lysis for Protein Isolation- URGENT - CORRECTION

Sergio sergioal at solea.quim.ucm.es
Tue Jan 18 07:00:00 EST 2000

I guess you don't have a french press, do you?

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.
> 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.
> Any help and advice, protocols greatfully accepted
> Many thanks
> Please reply directly to my email address:  m.d.jones at ic.ac.uk
> Dr Mick Jones, Lecturer in Molecular Virology
> Department of Infectious Diseases and Microbiology
> Imperial College School of Medicine
> Hammersmith Hospital, Du Cane Road, London, W12 0NN, UK
> Tel:  020-8383-3328;  Fax:  020-8383-2299
> email:  m.d.jones at ic.ac.uk
> -------------------------------------------
> "To Infinity and Beyond"  Buzz Lightyear (Toy Story)
> "Crackin' Toast, Gromit"  Wallace (The Wrong Trousers)
> ---

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