Polyacrylamide gel electrophoresis (PAGE) is one of the most frequently employed techniques for separating macromolecules including DNA, RNA, and proteins

Electrophoresis is in general the process of applying an electric field to move charged molecules through a solution. In this technique, the mobility of a charged molecule is directly proportional to its net charge and the resistance of the solution through which it is moving.

Sodium Dodecyl Sulfate (SDS) is an anionic detergent, and its molecules have a net negative charge within a wide pH range when dissolved. A polypeptide chain binds certain amount of SDS in proportion to its relative molecular mass, and the negative charges on SDS destroy most of the complex structure of proteins, especially after previous reduction with reagents such as DTT. Then the protein becomes a rod-like, negatively charge macromolecule with comparable hydrodynamic qualities, the mobility of which depends only on their molecular mass.

However, when a mixture of proteins is added onto the gel, the smallest of the proteins will start to make their way through the gel even before the entire sample has entered the gel. In order to achieve better separation, the entire sample is condensed into the narrowest possible band right at the interface of the gel through a neat trick called a stacking gel, and here is how it works.