Anti-Ubiquitin Mouse Monoclonal Antibody (Trial size)

AT

Signal-Seeker Ubiquitination Detection Kit (Cat. # BK161)
Signal-Seeker Ubiquitin Affinity Beads (Cat.# UBA01-beads)
Signal-Seeker: BlastR Rapid Lysate Prep Kit (Cat. #BLR01)

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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 ug of Swiss 3T3 cell lysate from cells treated for 5h with 10 uM MG132 (Lane 6) or untreated cells (Lane 7). Arrow indicates free ubiquitin band (8 kD), higher molecular weight bands are ubiquitinated proteins. To see the full Western blot protocol, see the product datasheet.
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.