gel electrophoresis - problem with stacking (Jan/18/2007 )
Hi, does anyone know why it can be happening this: When doing gels I have no problem with the polimeration of the gel from below but, the stacking keeps liquid or takes hours to be made. 
......it's really weird since I use exactly the same things that for the first one except for the buffer (pH 6.8 instead of 8.8).
Now I found a solution....I use the same pH 8.8 buffer for both parts and it works.......
I' don't know what might be happening, I'already checked the pH so that's not the reason.
By the way, do you know why the buffers have to have different pH?? 
well, hoping to find the reason!! 
You should increase the amount of temed (twice)
The different pH in the stacking gel is the trick of the system!!! At 6,8 the glycin in the running buffer is near its pI, therefore glycin will not run well in the gel, there is a lack of ions and your sampleproteins (loaded with SDS) will be focused in a disc. When you change the buffer in the stacking gel, you lose the system of focusing and than you can skip the stacking gel...
Maybe your TEMED is to old or your APS-solution is not fresh. We´re using 1% of APS-Solution (10%, fresh, 1-2 days) and 0,1% TEMED for the stacking gel.
Sometimes the stacking gel won't polymerize when you have traces of ethanol on top of your stacking gel. But I agree with Ms-Olli, I would make fresh APS and use also fresh TEMED.
it's a lab mith... if you keep your APS at 4ºC, it works for a long long time... of course I only make 10 ml, but I can finish it without polimerization problems in my PAGE...
it's a lab mith... if you keep your APS at 4ºC, it works for a long long time... of course I only make 10 ml, but I can finish it without polimerization problems in my PAGE...
thank you all for the info....
I'll see if I solve the problem
it's a lab mith... if you keep your APS at 4ºC, it works for a long long time... of course I only make 10 ml, but I can finish it without polimerization problems in my PAGE...
I made 10 ml of APS and aliquoted them into 500ul tubes, then stored them at -20degree.
Thaw it when need.
If the TEMED turns yellow, it has been oxidized by the moisture in the air and the strength decrease.
You can still use it, just increase the volume.
i always use fresh APS
other people here made fresh APS for each gel, however I store aliquots at -20ºC at it has always work fine for me.