Hello,

I do not know of glycerol in the separating gel. I would however feel the water or butanol overlay is important because it removes any airbubbles and leads to a uniform layer of separating gel which is very important. Your bands will not be straight otherwise. It also helps in faster solidification of the gel.

Cheers.

Hi Asa

You can use the method you mentioned but you need to be highly cautious. Water is a better option as you can be sure about the uniformity of  separating gel. The separating gel must be uniform for acceptable results. If separating gel is not uniform, you will get different results  each time and you will never know the exact reason. Therefore, I will recommend using water.

However, if you think you can be technically perfect in following all the steps of glycerol method, then go for it.

In case of native emsa gel recipes usually glycerol is used in the gel, which is expected to stabilize multiprotein complexes in the gel. So i would not expect problems if you add glycerol. But compared to butanol overlay, it will be technically hard to achieve a good stacking layer based on glycerol density in the gel mix. The degasing step could also be critical in such a case so that polymerisation is fast (we dont do that). If it works for you let us know.

Hi Asa,

Instead of buying gradient gel, you can make two different concentration of separating gels on the same gel and add 10% glycerol to the separating gel stock for the bottom separating gel. You still need to add water/butanol upon pouring the upper separating gel, before stacking gel. Hope this help!