SDS-PAGE --problems in resolving bands - (Jan/07/2008 )
So glad to have found a forum like this where everyone can help one another! I've been running some SDS-PAGE gels and use Invitrogen Benchmark prestained protein ladder on a 10% separating gel. Have been having a problem resolving bands beyond marker 6 (37kDa)-they just don't separate out. All markers larger than 37kDa separate nicely and it almost seems as if i am using a low percentage separating gel such that only the high MW markers get separated out well. But the strange thing is that when i use the resolving buffer made by my supervisor (and all other gel components the same) it separates out till marker 8 (19 kDa i think), so I'm convinced it's the resolving buffer that has a problem. I've made this buffer up twice (with the same recipe as my supervisor's) but with the same results-markers just resolving till 37 kDa. The next and last marker i can see runs ahead of the dye front, which is strange too. Does anyone have any suggestions as to what I can look at?
Another question-are sample bands in the gel supposed to run as a single line? Recently I've noticed that all my samples separate into 3 different shades of blue when they're running during SDS-PAGE...the whole dye front alone stretches about 4-5mm.
Please let my know if you have any answers/suggestions...Anything will be greatly appreciated, thanks!
Did you use the same SDS batch as the one used by your supervisor?
and other buffer components?
do you adjust the pH? does your supervisor?
do you use the same balance?
Thanks for your replies. Yes I did use the same batch of all the chemicals (Tris base and SDS), measured on the same balance. We both adjusted the pH to 8.8, and all chemicals were mixed thoroughly. I used a pH meter from another lab though and their HCl, but my supervisor has checked the pH of my buffer on the pH meter she used, and pH is fine. So all I can think of that is different is the HCl, although the other lab has been using this same HCl to make their resolving buffer, and have had no problems... so i can't really figure out what's going on...
the buffer is a stock? do you dilute correctly? mix well?
Do you let it polymerize long enough?
I dont know if this info is relevent.
For sds tank buffer my protocol says dont adjust pH with HCL.
When i added hcl to tank buffer i got bad separation of molecular weights.
The pH comes correct if components like tris and sds are weight correctly and if sds solution is used instead of directly adding sds powder to buffer.
Please correct me if i am wrong since i am new to this technique
you are correct. the buffer does not need to be adjusted if prepared carefully. you can check the pH and it should be around 8.3 but does not have to be exact. when you adjust the pH you add salt to the buffer, this will cause your problem.