I work in a lab were we have a common stock of 30% Acrylamide, TEMED, TRIS-HCl (pH6.8 and pH 8.8.) and a 10% (w/v) SDS solution pH 7.2.
We use the recipe for loading buffer (which we of course mix with BME before use):
- 3.15 mL MilliQ water
- 1.25 mL 0.5M Tris-HCl
- 2.9 mL 85% Glycerol
- 2 mL 10% SDS
- 0.4 mL 0.5% (w/v) bromophenol blue
Our problem is that there is no separation.
I have tried making 2 gels because I though I might have mixed the separating and the stacking gel, but I made sure i did not do this the second time. Then I asked someone else to make the gel, and it did not work for them either (nothing on the gel, no samples no ladder).
Then I remade the 10% SDS solution and both the TRIS buffers, but again something strange is going on as we get nothing on the gel.
The gels are obviously polymerized as its a solid gel when you take out to stain and destain, and I dont understand how the loading buffer can do this even if you mess it up (because you should see something on the gel as long as your sample in the well, even without denaturing). We obviously remade the loading dye and tried again, but this does not work.
I know there is protein in the samples, as I am putting cell pellet and purified protein (which I have tested the catalytic activity of) on the same gel.
Does anyone have a rational explanation for this, or suggestions of what can typically be wrong.
Bellow is a picture of the gel as it looks when its almost done running at 200V (80mAmp) for approx 40min. When we stain and destain there is nothing on the gel. Also I think the color (bromophenol blue) looks as if its running a bit strange on the gel, not as it normally should, see picture below, but perhaps I'm getting a bit paranoid.