I have a problem with PAGE SDS-electrophoresis of protein samples containing 0,5% of Triton X-100. Samples travel wery poorly and can't be properly stained (with Coomasie or silver - I get just smears). How should I solve the problem?
I used to work with membrane proteins and i really understand your problem. My protein did get stained by Coomassie blue, though. They did look funny on the Tricine SDS PAGE. Instead a smiling band, they were opposite. The smears you found on the gel may be due to degradation. Add proteinase inhibitors to your sample, such as PMSF to see if it makes difference
Triton X-100 can't be dialysed away. If you do need to remove the triton later, you can use Biobeads 5M2 (BioRad) to get rid of triton. The beads need to be washed by certain procedures before use. Add beads to your sample, mix for 30 min, spin and repeat until all detergent is removed.
I find the solution for my problem.
First I have prepared methanol:chloroform (2:1 v/v) solution and mixed it with sample in 1:1 ratio, vortexed, centrifuged (eppendorf centrifuge, 5 min at 13000 rpm) and removed upper (water) layer. After that I added pure methanol to sample (3:1 ratio) to precipitate proteins and centrifuged for 5 min at 13000 rpm, removed supernatant and washed precipitate once with 1 ml of methanol. Finally I suspended precipitated proteins in Laemmli sample buffer. Electrophoresis was fine after that :)
I’m using precast Bis-Tris gels from Invitrogen and time to time I have to load my samples dissolved in 2% Triton-X 100. I haven’t had any problem with the separation so far...
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