For some months we have had problems with SDS-PAGE polymerarization. We have changed the acrylamide/bisacrylamide solution, the TEMED, the APS but the problem continues. Does anyone have an explanation?
Thanks to all. It seems that the water was the problem. We have used new water obtained from other mili-q system and now it works well. Problably pH or salt concentration.
What kind of SDS gels do you use? (8%,10%,12%,...) Depending on this, the volume of APS and its molarity is important to let a gel polymerize completely.
We used different concentrations and we had problems with all of them. It is true that if we increase APS and TEMED amount, it works better, but it is not good enought. In addition, it varies from one gel to another.
Hi, Francisco,
What kind of polymerization problems do you have? The gel is not polymerized at all or it takes a longer time?
You can also try to use SPS instead of APS.
Hi, This is the protocol I follow for 2 minigel for the miniprotean , it works fine to me
Amount for two 10 % resolving gels / 1.5 mm )
8.2 ml distilled H2O
5 ml Tris-HCl 1.5 M, pH 8.8(BioRad)
6.5 ml acrylamide/bisacrylamide 30% solution (BioRad)
200 ul SDS 10%
mix gently.
Then add
100 ul APS
20ul TEMED
Mix quickly and pour into the glasses, and put butanol to cover the upper part). Let it polymerize for aprox 40 min. This will depend on the lab temperature (faster when it is hot) Then, wash the butanol out with water and pour the stacking gel (4%)
Amount for two 4% stacking gels.
6.1 ml distilled H2O
2.5 ml Tris-HCl 0.5 M, pH 6.8(BioRad)
1.33 ml acrylamide/bisacrylamide 30% solution (BioRad)
100 ul SDS 10%
100 ul APS
20 ul TEMED
mix and quickly pour over the polymerize resolving gel. Putt the combs and let it polymerize (about 15 min)
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