Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    Ubiquitin-AMC
    Catalogue Number:
    60-0116-050
    Size:
    50 µg
    Price:
    £200
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  • Species
    human
  • Source
    synthetic
  • Quantity
    50 µg
  • Storage
    -70°C
  • Concentration
    2mg/ml
  • Formulation
    DMSO
  • Molecular Weight
    8.72kDa
  • Stability
    12 months at -70°C; aliquot as required
  • Protein Sequence
    Accession number: P62987. C-terminally tagged with AMC
  • QA; Protein Identification
    Confirmed by mass spectrometry.
  • QA; Activity
    Activity Assay: The activity of Ubiquitin-AMC (7-amido-4-methylcoumarin) was validated by determining the increase in fluorescence at 460nm (Excitation 380nm). Increased fluorescence is a result of the enzyme catalysed cleavage between the C-terminal Glycine and AMC, creating Ubiquitin and de-quenched AMC. UCHL3 (deubiquitylase) was incubated with Ubiquitin-AMC and the fluorescence was measured at four time points (0min, 30min, 60min and 90min).

In addition to fusion proteins, ubiquitin derivatives conjugated with a fluorophore have been reported as substrates for biochemical DUB assays. A frequently used coumarin-based substrate is ubiquitin7-amido-4-methylcoumarin (Ub-AMC). DUBs catalyze the release of the AMC moiety, which is directly attached to the C-terminus of ubiquitin, and liberation of the fluorophore results in de-quenching of the fluorescent signal (Hassiepen et al., 2007). The excitation/emission range of this fluorophore is 380nm/460nm respectively. The use of this substrate for determining steady-state kinetic parameters in a number of DUB assays was first described by Dang et al. (1998).
References:

Dang LC, Melandri FD and Stein RL (1998) Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4methylcoumarin by deubiquitinating enzymes. Biochemistry 37, 1868-1879.
Hassiepen U, Eidhoff U, Meder G, Bulber JF, Hein A, Bodendorf U, et al. (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal Biochem 371, 201-207.