Hello,
I'm trying to run a NuPAGE Tris-Acetate 3-8% precast gel in reducing condition in order to detect a high molecular weight protein (360 kDa) by western blot.
I used the regular running buffer (Tris-Glycine with SDS) and regular loading buffer (with beta-mercapto), but something went wrong.
suggestions?
Dear Camilla. To answer your buffer question in the tile of your query yes you can (use Tris glycine running buffer)
What exactly went wrong ? Can you be more specific ?
Unfortunately you can't just use any running buffer, you have to use one compatible with your gel buffer, in this case a Tris-Acetate running buffer, otherwise your gel will not run properly. pH of gel and running buffer should be the same too, simple rules of conductivity apply!
Protein gels are carefully designed so that migrating ion zones contribute for better separation and/or formation of sharper bands. Therefore, ion compositions of gel and running buffer are very important. If you use wrong buffer, you cannot get full performance of the gel.
As for loading buffer, both SDS loading buffer (=Laemmli buffer) and LDS loading buffer seem to work.
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