I'm wondering how stable a master mix of SDS-PAGE ingredients (without APS and TEMED) would be when stored in a 4ºC laboratory refrigerator. The master mix would contain water, acrylamide, Tris buffer, and SDS; when we need to make gels, we would aliquot a certain amount to add TEMED and APS.
Hello Vincent,
Yes, when preparing SDS-PAGE gels, you don't add APS, TEMED as well as SDS to the pre-mix until just before casting. You may store the pre-mix at 4°C to prevent premature polymerization and degradation of the acrylamide monomer.
Why should you add SDS to the stacking and resolving gels?
SDS, a detergent, is used to unfold proteins and give them a negative charge, which is essential for separating proteins by size in SDS-PAGE. It is added to the sample buffer, and not to the gel.
SDS is added to the sample buffer in high concentrations to disrupt the non-covalent bonds within proteins, causing them to unfold and become negatively charged. The running buffer should also contain SDS to maintain a constant high concentration of SDS around the protein-SDS complexes throughout the electrophoresis process.
SDS is not necessary in the stacking and resolving gels because the proteins are already denatured and coated with a uniform negative charge before they enter the gels.
So, you prepare a pre-mix containing water, acrylamide and Tris buffer and store it at 4ºC. But I would not recommend making a pre-mix or master mix. You have to prepare the mix fresh, each time, which takes just a few minutes.
Best,
In my experience the acrylamide/tris mix is not stable it tends to degrade over time even in the fridge: I prefer to add each ingredient each time...
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