Antigen: native bovine ubiquitin conjugated to KLH · Host: rabbit · Application(s): WB, IP, and ChIP · Ubiquitin is a small protein that occurs in all eukaryotic cells. The ubiquitin protein itself consists of 76 amino acids and has a molecular mass of about 8.5 kDa. Key features include its C-terminal tail and the 7 Lys residues. It is highly conserved among eukaryotic species: human and yeast ubiquitin share 96% sequence identity.¹ The main function of ubiquitin is to clear abnormal, foreign and improperly folded proteins by targeting them for degradation by the 26S proteosome.² Ubiquitination represents an essential cellular process affected by a multi-enzyme cascade involving classes of enzymes known as ubiquitin-activating enzymes (E₁s), ubiquitin-conjugating enzymes (E₂s or Ubcs) and ubiquitin-protein ligases (E₃s). Ubiquitin is activated in a two-step reaction by an E₁ ubiquitin-activating enzyme in a process requiring ATP as an energy source. The initial step involves production of a ubiquitin-adenylate intermediate. The second step transfers ubiquitin to the E₁ active site cysteine residue, with release of AMP. This step results in a thioester linkage between the C-terminal carboxyl group of ubiquitin and the E₁ cysteine sulfhydryl group. The third step is a transfer of ubiquitin from E₁ to the active site cysteine of a ubiquitin-conjugating enzyme E₂ via a trans(thio)esterification reaction. And the final step of the ubiquitylation cascade creates an isopeptide bond between a lysine of the target protein and the C-terminal glycine of ubiquitin. In general, this step requires the activity of one of the hundreds of E₃ ubiquitin-protein ligases (often termed simply ubiquitin ligase). E₃ enzymes function as the substrate recognition modules of the system and are capable of interaction with both E₂ and substrate.^2,3 Ubiquitination also participates in the internalization and degradation of plasma membrane proteins such as some of the TCR subunits while still ER-membrane associated.⁴ Ubiquitin also plays a role in regulating signal transduction cascades through the elimination inhibitor proteins, such as IκBα and p27.⁵

1 Wilkinson, K.D. Roles of ubiquitinylation in proteolysis and cellular regulation. Annu Rev Nutr 15 161-189 (1995).

2 Bonifacino, J.S., and Weissman, A.M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev Biol 14 19-57 (1998).

3 . Ubiquitin Proteasome Pathway. (2009).

4 Yang, M., Omura, S., Bonifacino, J.S., et al. Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: Importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes. J Exp Med 187(6) 835-846 (1998).

5 Chen, Z.J., Parent, L., and Maniatis, T. Site-specific phosphorylation of IkBa by a novel ubiquitination-dependent protein kinase activity. Cell 84 853-862 (1996).

Chen, Z.J., Parent, L., and Maniatis, T. Site-specific phosphorylation of IkBa by a novel ubiquitination-dependent protein kinase activity. Cell 84 853-862 (1996).

Yang, M., Omura, S., Bonifacino, J.S., et al. Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: Importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes. J Exp Med 187(6) 835-846 (1998).

Bonifacino, J.S., and Weissman, A.M. Ubiquitin and the control of protein fate in the secretory and endocytic pathways. Annu Rev Cell Dev Biol 14 19-57 (1998).

Wilkinson, K.D. Roles of ubiquitinylation in proteolysis and cellular regulation. Annu Rev Nutr 15 161-189 (1995).

. Ubiquitin Proteasome Pathway. (2009).

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