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E. coli lysis for Protein Isolation- URGENT - CORRECTION

Michael Jones mick at 62wwc.freeserve.co.uk
Sat Jan 15 06:55:20 EST 2000


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
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