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My SDS-PAGE loading gets orange during the electrophoresis - (Oct/14/2008 )


I’m currently having a problem running my 12% SDS-PAGE gels. Sometimes, during the electrophoresis, my loading buffer (which has a blue colour) gets orange. When this happens, I don’t get a nice resolution of my proteins, especially for low weight proteins. What could be the problem?

My loading buffer’s recipe for 10ml of a 4X solution is:

- 2 ml Tris-HCl, 1 M pH 6.8
- 626 ul β-mercaptoethanol
- 4 ml SDS 20 %
- 4 ml glycerol 100 %
- 0.04 g bromophenol blue

The loading buffer stock solution is in fact orange, but when I dilute it to 1X it becomes blue. What is causing this effect during the gel, and how can this affects its resolution?

Bromophenol blue can get a yellow colour below pH 3.6, but what can cause this kind of pH alteration during my electrophoresis? Stacking gel has a Tris-HCl 0.5 M pH 6.8 buffer and separation gel has a Tris-HCl 1.5 M pH 8.8 buffer.

Thanks a lot,


Your stock solution should not have such an orange color, there is something wrong with your pH. Measure your solutions when you preprare the stock solution, perhaps your Tris-HCl is in fact much more acidic. The same is true for your gel solutions, measure their pH and see if it is still allright. Also your running buffer might add to this effect, look at its pH as well.

The resolution is dependant on the right pH gradients between stacking and resolving gel, make sure, that everything is fine with it, best to prepare fresh solutions.


some people make fresh SDS-PAGE buffer once in a while to solve such problems.


Your loading buffer stock should be blue instead of orange... too high pH.

may check the pH of your stock with a pH paper.

When you diluted your loading buffer, the buffer changed from orange to blue...This may be because the dilution with a solution decreased the pH of the buffer, and changed it into blue (marginally)...

During the electrophoresis, the marginally blue loading buffer changed back into orange due to pH fluctuation.

Hope this may help.

-Minnie Mouse-