What is a separating gel used for?

In the SDS-PAGE technique, the high concentration separating gel is placed at the bottom of the gel electrophoresis chamber, below the low concentration stacking gel, which has larger pores. The main purpose of the separating gel is to slow down the migration of proteins so that all proteins enter the separating gel at the same time.

When the proteins from the stacking gel flow down and reach the separating gel, the smaller pore size of the separating gel acts as a barrier, slowing down their migration. The slower migration rate allows other slower migrating proteins to catch up creating a narrow, concentrated, and distinctly visible band in between the two gels. The separating gel separates molecules based on size and charge.