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Ladder and proteins do not migrate into stacking gel - (Aug/06/2014 )

We're having a problem in our lab I haven't seen before. We're pouring standard Tris-glycine SDS-PAGE gels with the pre-made acrylamide/bisacrylamide from Bio-Rad. The protein samples, as well as the ladder, are not migrating properly into the resolving gel from the stacking gel: fragments smaller than 100 kDa do enter the resolving gel while larger size fragments do not. The stacking gel is at the standard 4% and we checked the pH of the Tris buffers used to make the stacking and resolving gels. Does anyone have a suggestion? It's not just a sample problem, as the ladder is doing this also!

-ASL-

What % resolving?

-bob1-

The resolving gel is 10% (not anything crazy high).

-ASL-

Hmmm. Make some fresh SDS and try again.

-bob1-

or were you mixing the buffer for SDS-PAGe right as a protocol.? 

-Miaaa-

Hmmm. Make some fresh SDS and try again.

use a different lot of sds

 

also

 

did you prepare the buffers or someone new to the lab?

 

did you check the pH of the sample buffer?

 

how old are the components of the sample buffer?

 

did you overboil your samples?

 

has it happened more than once?

-mdfenko-