Ubiquitin-Rhodamine 110

Ubiquigent

Catalog No: UBI-60-0117-050

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Species Reactivity: human  
Source: synthetic  
Formula: DMSO  
Concentration: 2mg/ml  
Molecular Weight: 8.93kDa  
Protein Sequence: Accession number: P62987. C-terminally tagged with Rhodamine 110  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control 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.