Ubiquitin-AMC

Ubiquigent

Catalog No: UBI-60-0116-050

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Species Reactivity: human  
Source: synthetic  
Formula: DMSO  
Concentration: 2mg/ml  
Molecular Weight: 8.72kDa  
Protein Sequence: Accession number: P62987. C-terminally tagged with AMC  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control 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).  

Background

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.