Michael Jones wrote:
>> 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
>> 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
>> 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.
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