I am using 29:1 Acrylamide/Bis-acrylamide solution to prepare 12 % resolving gel and 4% stacking gel. The pH of the resolving and stacking gel buffers are pH 8.8 and pH 6.8 respectively. after running the gel, I could see a transparent line across the width of the gel corresponding to 16 kDa and the ladder (6.5 kDa) is after that. That transparent line ran behind the dye front. After staining, I could see that transparent line stained and the proteins are restricted by that. What might be the reason? will prerunning the gel help, so that the transparent line will run off? How does the prerunning of the gel affects the discontinuous buffer system?
I was facing the same problem as your mention. and it was found protein band under the refractive line was not sharp when stained by silver staining. I fix it by pre-running my gel for 1 h and then I obtained the protein band sharply. If you do not want concentrate your sample in stacking gel (Because function of stacking gel can be lost by pre-running). you can perform pre-runing the gel to get rid of refractive line.
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