The zone where this effect appears is not corresponding with the stacking gel zone, no? Have your buffers been used for long time? These kind of effects some times disappear by changing all the buffers and even the acrylamide bottle, if it was used for a long time. If you have not done so already, you can repeat the assay with new buffers and acriylamide.

Thank you María Luisa Caballero for your response.

No; it is a separating gel zone.

I'll try as you suggest.

Thank you again.

I still don't know the exact problem.

But I changed the running buffer and it worked perfectly.

THANK YOU María Luisa Caballero.