In article <sf211260.089 at VETMED.TAMU.EDU> GSONG at VETMED.TAMU.EDU (Guobin Song) writes:
>From: GSONG at VETMED.TAMU.EDU (Guobin Song)
>Subject: Non-denaturing SDS-PAGE
>Date: 21 Jan 1995 11:53:16 -0800
>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
You can try small diameter (0.35mm) glass beads. I've used them at different
scales (from small volumes in Eppendorfs to large volumes in PVC centrifuge
bottles). Generally you want as high a resuspended cell density as possible
plus the same volume of beads. Vortexing the tubes for about three to five
minutes in 30 sec. intervals allowing 1min for cooling on ice usually does the
trick. You could also try several freeze-thaw cycles on your cells.
Alex.
