Electrophoresis question

[Dymoon]

I'm a student and I'm doing enzymatic visualization in native electrophoresis, and the colored reaction works when i insert the gels in the substrate. The problem is that the migration is very slow with very tight bands with diffusion appearing sideways like they united.

Other weird thing is that the amperage is very small at 4-5 mA, while the voltage was set to 200V. Anyone met this situations before or now why they might happen?

[mchlbrmn]

I don't actually know what you are doing, or what kind of electrophoresis this is, but if the current (amps) is low, and it moves slower than normal, while the voltage is high it sounds like the salt concentration, or the general gel buffer concentration, is too low. It is the salt ions that carries the current, so if someone made the buffer too weak, or forgot to use any, things will not move much.

[Dymoon]

It's with Acrylamide-Bis-Acrylamide and tris-HCl buffer 6.8 stacking and 8.8 separation without SDS and the migration buffer is glycerol-tris . Sorry i forgot to mention.
Can the tris go old? Anyway thank you, i'll try remaking the buffers.

[mchlbrmn]

I don't think that Tris goes bad, unless it's not sterile and something grows in it, or if it's allowed to evaporate and the pH changes.
I don't have experience with that buffer for DNA, I think I used TBE. I would have thought salt would be added, but maybe there's enough salt in the Tris from when it's pH'ed.
Good luck.

[mdfenko]

It's with Acrylamide-Bis-Acrylamide and tris-HCl buffer 6.8 stacking and 8.8 separation without SDS and the migration buffer is glycerol-tris . Sorry i forgot to mention.
Can the tris go old? Anyway thank you, i'll try remaking the buffers.

that should be tris-glycine. it would explain your problem if you used glycerol.

if not then you should ensure that you use the chemical grade(s) called for in your protocol. and that you weigh and suspend carefully or your pH and concentration may be off.