IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

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.
>
> 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)

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






More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net