I do peptide western blot, with Tricine SDS PAGE, and try to separate 4.7 kDa and 5.1 kDa proteins. I use my own mini gel: 16.5% separating, thin layer of 10%, and 4% stacking gel. I usually run the gel overnight at 4C with 30V. I finally managed to separate two bands, but the U shape of the bands is very annoying. In high molecular weight regions I see even bands with coomassie staining. It must be to do with running conditions.
I think 10%seperating for such low molecular weight is not working.
Try higher percentage gel. I would suggest 15% or 16% seperating. Sometimes the bands at extreme lower side of the gel gives smileys. I would also check my running conditions like fresh running buffer, 6X sample buffer etc. one more suggestion is to run it on lower voltage try 100v or 120v.
Hope this will help. All the best. :)
Thank you for your quick answer! I use mini gel which has 3 parts: stacking 4%, intermediate layer 10% and a larger separating gel 16%. As I wrote I use 30V voltage for running, I think it is low enough but maybe It is much too low... Fresh sample buffer seems to be a good idea! Thanks!
- Badges
- Science method