Anti-ubiquitin-HRP antibody is a pan-ubiquitin, mouse monoclonal antibody conjugated with HRP that is part of the Signal-Seeker™ product line. The antibody was raised against full length bovine ubiquitin. The antibody has been shown to recognize poly-ubiquitin, mono-ubiquitin, and free ubiquitin (Fig 1). Ubiquitin is a highly conserved protein and AUB01 is predicted to recognize ubiquitin from a wide range of species (5). AUB01-HRP has been validated in western blot applications and has been shown to be more potent than unconjugated AUB01 used with standard mouse-HRP secondary. AUB01 is purified by Protein G affinity chromatography and is supplied as a lyophilized white powder.
Western Blot using Ubiquitin Antibody
Anti-Ubiquitin Antibody (Cat. # AUB01) was used at a 1:500 dilution. Bovine thymus ubiquitin was run as follows; Lane 1-50 ng, Lane 2-25 ng. Lane 3– 12.5 ng, Lane 4-6.25 ng, Lane 5-3.12 ng. Lanes 6 & 7 represent 20 µg of Swiss 3T3 cell lysate from cells treated for 5h with 10 µM MG132 (Lane 6) or untreated cells (Lane 7). Arrow indicates free ubiquitin band (8 kD), higher molecular weight bands are ubiquitinated proteins.
Western Blot using Ubiquitin-HRP Antibody
Untreated and MG-132 treated cell lysate were separated by SDS-PAGE and transferred to PVDF. Western blot using an unconjugated (AUB01, 1 µg/ml) or HRP-conjugated (AUB01-HRP, 1 µg/ml) ubiquitin antibody was performed. Anti-Mouse HRP secondary was used for AUB01 but not AUB01-HRP.
Ubiquitin (Ub) and ubiquitin-like proteins (Ubls, e.g. SUMO, Nedd) are a group of approximately 15 proteins that have a molecular weight of around 8 kD. During the ubiquitination process, these are conjugated via activating (E1), conjugating (E2) and ligating (E3) enzymes to lysines of a target protein (1). Mammalian cells express over 600 potential ubiquitin ligases which exceeds that of the kinase superfamily of PTM proteins (2).
One function of ubiquitination is to target proteins for proteosomal degradation. This role can range from a general housekeeping function that clears miss folded proteins from a cell to involvement in tightly regulated spatio-temporal cell signaling events (1). An emerging function of ubiquitination is its ability to activate proteins via the creation of unique protein:protein interactions (3). In common with many other PTMs, ubiquitination is reversible. Ubiquitin-specific proteases (USPs or DUBs) remove ubiquitins from target proteins (4). The reversible nature of ubiquitination further enhances the potential of this PTM to dynamically regulate protein function.
1) Grabbe, C. et al. 2011. The spatial and temporal organization of ubiquitin networks. Nat. Rev. Mol. Cell. Biol. 12:295-307.
2) Deshaies R.J. & Joazeiro, C.A. 2009. RING domain E3 ligases. Ann. Rev. Biochem. 78: 399-434.
3) Lomeli, H. & Vazquez. 2011. Emerging roles of the SUMO pathway. Cell Mol. Life Sci. 68:4045-4064.
4) Faesen, A.C. et al. 2012. The role of UBL domains in ubiquitin specific proteases. Biochem. Soc. Trans. 40:539-545.
5) Zuin, A. et al. 2014. Ubiquitin signaling: Extreme conservation as a source of diversity. Cells 3:690-701.