In article <sf211260.089 at VETMED.TAMU.EDU>, GSONG at VETMED.TAMU.EDU (Guobin Song) writes:
> Hello! I'm going to run some 35SMet-labeled Brucella
> (Gram-negative) whole cell proteins on non-denaturing
> SDS-PAGE. In order to keep the proteins at non-denature state I can't use SDS and other detergent to lyse the
> cells, I know I can sonicate the cells, but that would be my
> last resort, because I don't want to contaminate our
> sonicator with 35S. So is there any other method availabel
> that can lyse the cells? Any other suggestions on non-
> denaturing SDS-PAGE would be appreciated too. Thanks!
>> Guobin Song
> Dept. Vet. Pathobiology
> TAMU, TX
> email: gsong at vetmed.tamu.edu
>I think you can try to use glass beads (size of 40mesh or smaller). Just
put the sterilised (if necessary) glassbeads into the cell paste up to the
miniscus and vortex the tube for 20 seconds, cool (to prevent excess heat
formation) and repeat the vortex for a couple of times. Wash the glassbeads
cell debris with buffer and spin the collected lysate for a clear lysate.
Analyse the protein concentration and load sample on PAGE. Try this on non-
labelled cells first so you know whether it works or not. I have use this
method on E. coli before and it works.
