polyacryl polymerization in 10% glycerol - (Nov/23/2009 )
To anybody who could help;
I am trying to prepare a native PAGE gel for EMSA, where the gel contains 10% glycerol. I have tried to polymerize acrylamide (5% at 60:1 acyl:bis acryl) in Tris/Glycine buffer with 10% glycerol and cannot get it to polymerize, even though a 5% acrylamide gel in Tris/Glycine polymerizes readily.
I once tried to take a 5% gel polymerized in Tris/Glycine buffer (no glycerol) and pre-run it with running buffer with 10% glycerol before running the samples. However, when I did this, the film from the EMSA of this gel suggested that the samples smeared out. I question whther the glycerol, an uncharged molecule, was able to run through the gel under the voltage applied.
Any advice?
StevieRay on Nov 23 2009, 02:21 PM said:
I am trying to prepare a native PAGE gel for EMSA, where the gel contains 10% glycerol. I have tried to polymerize acrylamide (5% at 60:1 acyl:bis acryl) in Tris/Glycine buffer with 10% glycerol and cannot get it to polymerize, even though a 5% acrylamide gel in Tris/Glycine polymerizes readily.
I once tried to take a 5% gel polymerized in Tris/Glycine buffer (no glycerol) and pre-run it with running buffer with 10% glycerol before running the samples. However, when I did this, the film from the EMSA of this gel suggested that the samples smeared out. I question whther the glycerol, an uncharged molecule, was able to run through the gel under the voltage applied.
Any advice?
Well, thanks to anybody who considered my question.
It turns out what you need to do is put the acrylamide/buffer/glycerol/APS solution in a flask and degas it. While regular acrylamide solutions without glycerol do not need to be degassed for polymerization to occur, you MUST degas to allow polymerization in 10% glycerol
StevieRay on Nov 23 2009, 05:21 PM said:
yes, sort of. eof (electroosmotic flow, one of the main driving forces in capillary electrophoresis) will cause the glycerol to flow through the gel with the buffer, but eof is pretty weak in this situation.
degassing helps speed polymerization in all cases by removing dissolved oxygen (oxygen inhibits the free radical polymerization reaction). when mixing (vigorously?) the glycerol with the rest of the solutions you probably introduce a lot of oxygen.