Ubiquitin (Ub) is a 76 amino acid protein widely expressed in the cytoplasmic and nucleus of cells. Ub is posttranslationally conjugated to proteins by the E1, E2, E3 protein ubiquitination cascade. Ub can be conjugated on proteins as monoUb or polyUb chains. Protein ubiquitination plays both proteolytic and nonproteolytic functions. Usually, polyubiquitinated proteins are targeted to the 26S proteasome for proteolysis. Typical concentration to support in vitro ubiquitination is 50-100 μM. In Ub(K63 only), all lysine residues but lysine 63 were substituted with arginines, thereby it only supports the formation of lysine 63-linked polyubiquitin chains.
