The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Activity: Ki = 2.5 nM versus Isopeptidase-T.
Add directly to in vitro assay from the stock solution. Depending on conditions, typical concentrations to fully inhibit UCHs in vitro are 2-5 µM.
A potent and specific inhibitor of all ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs) and deubiquinating enzymes (DUBs). This protein blocks the hydrolysis of poly-ubiquitin chains on substrate proteins in vitro and thus enhances poly-ubiquitin chain accumulation.
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Preparation and Storage
Stability and Storage
Shipped at 4°C. Upon delivery aliquot. Store at -80°C. Avoid freeze / thaw cycle.
This product is an active protein and may elicit a biological response in vivo, handle with caution.
ReconstitutionSoluble in aqueous buffer up to 50 mg/ml. For best results, pellet dry
compound prior to reconstitution. Store powder at -20°C. Store stock solution at -80°C.
FunctionUbiquitin: Exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked: Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in lysosomal degradation; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling.
Sequence similaritiesBelongs to the ubiquitin family. Contains 3 ubiquitin-like domains.
Post-translational modificationsUbiquitin: Phosphorylated at Ser-65 by PINK1 during mitophagy. Phosphorylated ubiquitin specifically binds and activates parkin (PARK2), triggering mitophagy (PubMed:24660806, PubMed:24751536, PubMed:24784582, PubMed:25527291). Phosphorylation does not affect E1-mediated E2 charging of ubiquitin but affects discharging of E2 enzymes to form polyubiquitin chains. It also affects deubiquitination by deubiquitinase enzymes such as USP30 (PubMed:25527291).