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E. coli cell lysis for Protein Isolation URGENT HELP PLEASE

Scott Craig s.craig at student.unsw.edu.au
Mon Jan 17 20:20:20 EST 2000

Hi Mick,
If all you want to do is have a look at the total cell extract on a gel
then you can spin down the cells and lyse the pellet in SDS/PAGE loading
buffer and heat it up as would for any sample. Spin the sample after the
heating and it will help pellet some the DNA which can cause a bit of a
problem with it's viscosity. The protocol which comes with the maltose
binding protein system from NEB suggests 100ul of dye per 0.5ml of culture.

Alternatively if you want to get a lysed sample you can do things with,
Pierce have a detergent based lysis buffer called Bper which you resuspend
the cell pellet in a it busts open the cells which might be an easy option.

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.
> 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)

Scott Craig
Dept. of Biotechnology
University of New South Wales
Sydney, Australia

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