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:
>> 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
>> 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.
>> 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)
Dept. of Biotechnology
University of New South Wales