Detection and analysis of FAT10 modification.

FAT10 plays a role in many cellular processes. Nevertheless, only one substrate could be identified so far to which FAT10 becomes covalently attached via a nonreducible isopeptide bond. The identification of additional substrates as well as interaction partners is therefore of great interest. Due to the absence of potent anti-FAT10 antibodies, the detection and identification of interaction partners was hindered so far. We have recently described the generation of a novel monoclonal FAT10 antibody that is suitable for immunoprecipitation of endogenous FAT10 and describe in this chapter the detection of endogenous as well as of His-3×FLAG-tagged FAT10 interacting proteins and conjugates by immunoprecipitation using either this novel anti-FAT10 antibody 4FI or an anti-FLAG affinity gel.