Ubiquitin is a 76 amino acid (aa) protein that is ubiquitously expressed in all eukaryotic organisms. ubiquitin is highly conserved with 96% aa sequence identity shared between human and yeast ubiquitin, and 100% aa sequence identity shared between human and mouse ubiquitin. In mammals, four ubiquitin genes encode for two ubiquitin-ribosomal fusion proteins and two poly-ubiquitin proteins. Cleavage of the ubiquitin precursors by deubiquitinating enzymes gives rise to identical ubiquitin monomers each with a predicted molecular weight of 8.6 kDa. Conjugation of ubiquitin to target proteins involves the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and a lysine residue in the target protein. This process of conjugation, referred to as ubiquitination or ubiquitylation, is a multi-step process that requires three enzymes: a ubiquitin-activating (E1) enzyme, a ubiquitin-conjugating (E2) enzyme, and a ubiquitin ligase (E3). ubiquitination is classically recognized as a mechanism to target proteins for degradation and as a result, ubiquitin was originally named ATP-dependent Proteolysis Factor 1 (APF-1). In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as signal transduction, endocytosis, and post-endocytic sorting. Mature ubiquitin has a highly conserved C-terminal diglycine motif which is crucial for activity and recognition with conjugation and deconjugation enzyme components. The replacement of the last glycine residue with alanine results in a ubiquitin that supports ubiquitin-activating (E1) enzyme-ubiquitin thioester formation and downstream conjugation reactions (transfer to E2, E3) but at a rate ~20% compared to wild type ubiquitin.