Novel antibody tags from the rat lysosomal protein RT1.DM for immunodetection of recombinant proteins

Previously, two mouse monoclonal antibodies (12B8 and 6D4) were raised against the α- and β-subunits of the rat non-classical MHC class-II heterodimer RT1.DM. Here, I describe the epitope mapping of mAb 12B8 to amino acids α3-14 (EASPQAWWDESQ) and mAb 6D4 to amino acids β35-44 (WDPEEGQIVP). Epitope mapping was conducted by preparing fusion proteins between the α and β chain of RT1.DM for Western detection with mAb 12B8 and 6D4. By mutating non-conserved amino acids of the human orthologue of RT1.DM, the rat epitopes were introduced, thereby making the α and β polypeptides sensitive for mAb 12B8 and 6D4 detection. The epitopes, designated as 12B8 and 6D4, were tested for protein tagging. They were appended to the N- or C-terminus of four human proteins, the tumour suppressor protein VHL (von Hippel-Lindau), SUMO4, MHC class-II DQβ and -DPβ for expression in mammalian cells. Western detection, immunoprecipitation and localisation of the tagged proteins were successfully demonstrated. Thus, the 12B8 and 6D4 epitope tag can be universally used for the immunodetection of recombinant proteins and to study protein-protein interactions.