Enabling Protein Degradation Drug Discovery

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  • Name
    Catalogue Number
    Size
    Price
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  • Name:
    Ubiquitin-Rhodamine 110
    Catalogue Number:
    60-0117-050
    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
    8.93kDa
  • 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).

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.