How does a stacking gel work?

The stacking gel “stacks” proteins based on the low polyacrylamide content and low pH.

The large pore size derived from the low polyacrylamide content allows for freer movement for the proteins, giving a change for the larger ones and smaller ones to equalize with each other.

The low pH (6.8), on the other hand, allows glycine in the running buffer to be in its zwitterionic state. Comparing to the free Cl- ions (from Tris-HCl in the gel), these glycine zwitterions move slowly toward the anode due to the lack of charge. A narrow but steep voltage gradient is thus created between the Cl- ions (leading ions) and glycine zwitterions (trailing ions), with protein samples moving in between these two boundaries. As a result, the glycine keeps pushing the protein towards the Cl- ion, forming a tight band in the stacking gel.