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.
>> 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.
>> 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)
>> ---
