After IEF the second-dimension separation can be performed on various flatbed or vertical systems.

SDS-PAGE consists of four steps:

(1) Preparing the second-dimension gel,
(2) Equilibrating the IPG strip(s) in SDS buffer,
(3) Placing the equilibrated IPG strip on the SDS gel, and
(4) Electrophoresis.

In this guide the equilibration step is described first because it is a protocol common to both vertical and flatbed systems. Gel preparation, IPG strip placement, and electrophoresis protocols, on the other hand, are specific to the orientation of the gel. Note however, that the second-dimension gel must be prepared before the equilibration step is started.

SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is an electrophoretic method for separating polypeptides according to their molecular weights (MW). The technique is performed in polyacrylamide gels containing sodium dodecyl sulphate (SDS). The intrinsic electrical charge of the sample proteins is not a factor in the separation due to the presence of SDS in the sample and the gel. SDS is an anionic detergent that denatures proteins by wrapping around the polypeptide backbone in a ratio of approximately 1.4 grams SDS per gram protein. The bound SDS masks the charge of the proteins themselves, forming anionic complexes with constant net negative charge per unit mass. The SDS also disrupts hydrogen bonds, blocks hydrophobic interactions, and partially unfolds the protein molecules, minimizing differences in molecular form by eliminating the tertiary and secondary structures. The proteins are totally unfolded when a reducing agent such as DTT is employed. The disulphide bonds, which can form between cysteine residues, are cleaved, and the polypeptides become flexible rods of negative charges with equal "charge densities," or charge per unit length. When proteins are treated with both SDS and a reducing agent, separations exclusively by molecu lar weight are possible. In fact, there is an approximately linear relationship between the logarithm of the molecular weight and the relative distance of migration of the SDS-polypeptide micelle. (Note: This linear relationship is valid only for a certain molecular weight range that is determined by the polyacrylamide percentage.)