E. coli cell lysis for Protein Isolation URGENT HELP PLEASE

S Hopkirk s.hopkirk at virgin.net
Sat Jan 15 03:59:36 EST 2000


You can re-suspend the cells in a cell lysis buffer containing lysozyme.
The New England BioLabs recommend this  solution in their IMPACT system:
50mM Tris-HCl, 10mM NaCl, 10mM EDTA with only 10-20ug/mL lysozyme  to
prevent  excess viscosity due to chromosomal DNA.

Dr Mick Jones wrote:

> Hi
>
> 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 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.
>
> 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
>
> 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
> Tel:  020-8383-3328;  Fax:  020-8383-3394
> (Note new London code)
> 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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