In continuous systems, molecular charge density and gel pore size are the only factors that have any effect on the stacking, or zone
sharpening, of a molecule. The proteins in a sample applied to a continuous system will move faster in the sample well than in the gel.
A concentrated band of sample will form where the molecules are slowed down at the interface of the buffer and the restrictive gel.
This type of stacking has limitations, particularly when separating molecules which vary widely in size. The smaller molecules have
less of a difference between their free solution mobility and their mobility in a gel so the sharpening factor is not as favorable as for the
larger molecules.