Analysis of modification and proteolytic targeting by the ubiquitin-like modifier FAT10

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.