Is my gel not polymerising fully and why......? - (Apr/28/2005 )
Hi, I run a 11% (pH 8.8) seperating gel and a 4% stacking gel (ph 6.8) and pour my own gels for the Biorad mini-Protean three system. After finally optimising all my anitbodies and blockings my gels are now all running 'funny' below 66kDa. I have perfectly straight bands above this point (non-specific staining) but where I want to see my band all the bands and markers have merged into each other in a big wave-like fashion.
Everything's fresh, pH's are good, I'm waiting an hour before pouring my stacking gel and another hour before running. Left over gel has certainly set and the top half of the gels are fine - WTH is going on with the bottom????? - oh and we can't afford pre-poured gel so don't suggest that as a solution!
did you check the polymerisation of your gel? You could disassemble your poured gel and touch it. But there should be no reason why it polymerises in the upper part but not in the bottom.
Maybe you have to much salt in your sample?
I do not wait so long after pouring my gel: 10min for my seperating gel and 20min for my stacking gel. I use 100µl 10%APS/10ml gel and 10µl TEMED/10ml gel.