Immunoblotting of Peroxisomal Proteins with Monospecific Antibodies

Abstract

The most commonly used protein electrophoresis system today is the method described by Laemmli [2]. The Laemmli procedure, named sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), is a discontinuous system: the pretreated proteins are first concentrated in a stacking gel before entering the separating gel. Before electrophoresis, proteins are denaturated using SDS, an anionic detergent, that wraps around the polypeptide backbone and confers a negative charge to the polypeptide in proportion to its length. Proteins are also treated with a reducing agent such as 2-mercaptoethanol, which breaks disulfide bonds. After treatment, polypeptides are separated on the support gel, a Polyacrylamide matrix, on the basis of molecular weight by means of an electrical field in the presence of SDS. The major use of this system is to determine the molecular weight of polypeptides by running the gel silmutaneously with standard polypeptides of known molecular weights. A linear relationship exists between the log₁₀ of the molecular weight of a polypeptide and its Rf (the distance from the limit between the stacking and separating gels to the polypeptide band, divided by the distance from the limit of the two gels to the front underlined by a low molecular weight dye).