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 use a procedure for E. coli that involves suspension in a sucrose solution,
then sequential treatment with lysosyme, EDTA, and Triton X-100. This works
well for small volumes of cells (1 ml of culture => 200 micoL of lysate) - I
haven't tried it for larger volumes, but it should work... The reference is
Clewell and Helinski (1969) Proc Natl Acad Sci USA, 69, 2110-2114.
Whether this will work for your bugs, I don't know!
[You could off course use a sonicator and clean the tip (with eg CountsOff then
lots of water) afterwards]
Good luck,
Richard.
P.S. I hate to be pedantic, but..You can have ND-PAGE or SDS-PAGE, but not
ND-SDS-PAGE!
