Ubiquitin-AMC


Catalogue Number
60-0116-050
Product Size
50 µg
Price £
£200
Accession Number
P62987
Residues Expressed
n/a
Certificate of Analysis Size
50 µg
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).


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.