Ubiquitin-Rhodamine 110


Catalogue Number
60-0117-050
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
8.93kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: P62987. C-terminally tagged with Rhodamine 110
QA; Protein Identification
Confirmed by mass spectrometry.
QA Activity

Activity Assay: The activity of Ubiquitin-Rhodamine 110 was validated by determining the increase in fluorescence at 535nm (Excitation 485nm) measured as a result of the enzyme catalysed cleavage at the amide bond between the C-terminal Glycine and Rhodamine, generating Ubiquitin and de- quenched Rhodamine 110-Glycine. UCHL3 (deubiquity-lase) was incubated with Ubiquitin-Rhodamine 110 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. Ubiquitin-Rhodamine 110 (Ub-Rho110-G) is a fluorogenic rhodamine-based substrate. While the disubstituted rhodamine moiety in UbRho110-G is essentially non-fluorescent, cleavage results in a mono-substituted rhodamine, Rho110-G, which exhibits intense fluorescence when excited at 485 nm (Hassiepen et al., 2007). The rhodamine fluorophore exhibits optical properties more appropriate - than Ubiquitin-AMC – for compound screening and profiling. The risk of artifacts in screens due to autofluorescence of compounds is substantially reduced as the rhodamine 110 fluorophore has excitation and emission wavelengths of 485nm and 535nm respectively (Hassiepen et al., 2007).


References

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.