Originally Posted by butters
have you check the pH of your resolving and stacking buffer? Are you still using the same buffer for the pass 6 month? Have you make new stock to try?
Is the gel took longer to polimerize? Or the gel feel less firm than previously? What is the percent of gel did you prepare? You could prepare fresh ammonium persulfate. unless your ammonium persulfate powder is "kinda" wet.
Can you please list out what reagent change and you still experiencing this problem.
Check the pH of the buffers for making the gel as well as the running buffer. Make sure that the stacking and resolving buffers haven't been switched around. Making the resolving gel with stacking gel buffer could create this problem because the proteins would not "de-stack".
4x Stacking buffer (upper gel) should be:
0.5M Tris, pH 6.8
4x Resolving Buffer (lower gel) should be:
1.5 M Tris, pH 8.8
This looks like a major problem so I don't think it's some thing that is a little off, it must be something seriously messed up.
If you make your own running buffer (tris-glycine-sds) remember NEVER EVER pH this with HCl. The buffer does not need to be pH-ed, if the correct amount of TRis and Glycine are added, the pH will be ok. This will make your gel run very strangely (usually you notice this because the voltage/current is way off from what it normally is).