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 Ubiquitinribosomal

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 multistep 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 (APF1) .

In addition to protein degradation, ubiquitination has been shown to mediate a variety of biological processes such as

signal transduction, endocytosis, and postendocytic sorting . This Nterminal HAtagged Ubiquitin protein allows for the

convenient detection or affinity purification of ubiquitinated proteins in vitro. The HA peptide sequence

(YPYDVPDYA) is an epitope derived from the influenza Hemagglutinin protein. This tag is specifically recognized by

anti HA antibodies and anti HA agarose.