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Ladder and proteins do not migrate into stacking gel

SDS-PAGE issues protein migration

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5 replies to this topic

#1 ASL

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Posted 06 August 2014 - 10:11 AM

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!



#2 bob1

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Posted 06 August 2014 - 12:56 PM

What % resolving?



#3 ASL

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Posted 07 August 2014 - 09:14 AM

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



#4 bob1

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Posted 09 August 2014 - 01:53 PM

Hmmm. Make some fresh SDS and try again.



#5 Miaaa

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Posted 10 August 2014 - 08:32 PM

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



#6 mdfenko

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Posted 11 August 2014 - 06:25 AM

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?


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genius does what it must
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