Product Datasheet
QW-002
Ub-Rho110 is a high-sensitivity fluorogenic substrate with ubiquitin C-terminus covalently conjugated to Rhodamine 110. Compared to the classic Ub-AMC substrate, Rho110 offers significantly higher quantum yield, superior photostability, and longer excitation wavelength, effectively minimizing autofluorescence interference from biological samples. It is the ideal choice for DUB inhibitor high-throughput screening (HTS) and low-abundance enzyme activity detection.
In the intact Ub-Rho110 molecule, ubiquitin moiett occupies amino groups of Rhodamine 110, resulting in complete fluorescence quenching. Upon DUB cleavage, free Rho110 is released, producing strong fluorescence at Ex 485 nm / Em 528 nm in the visible spectrum, compatible with standard fluorescence plate readers and microscopes.
Recommended final substrate concentration: 0.1–1 μM. Buffer: 50 mM HEPES (pH 7.5), 0.5 mM TCEP, 0.1 mg/mL BSA. TCEP is preferred over DTT for Rho110 systems to avoid DTT-induced non-specific fluorescence.
| Cat. No. | QW-002 |
| Molecular Formula | C₄₀₀H₆₄₄N₁₀₈O₁₂₁S |
| Purity | ≥95% (HPLC) |
| Specification | 1mg / 5mg / 定制 |
| Ex | 485 nm |
| Em | 528 nm |
| Storage | -80°C 避光保存 |
RP-HPLC-MS was employed to analyze Ub-Rho110. The measured molecular weight is in excellent agreement with the calculated theoretical value of 8,933.9 Da.
Reversed-phase HPLC was performed to assess the purity of Ub-Rho110. Welch® XB-C18 column (4.6×250 mm, 5 μm), flow rate 1.0 mL/min, detection wavelength 214 nm. Purity ≥ 95%.
SDS-PAGE was conducted to verify the molecular weight and purity of Ub-Rho110. The Coomassie Brilliant Blue R-250 staining result is shown below.
50 μg Ub-Rho110 was dissolved in DMSO and diluted to 280 μL (20 μmol/L) in assay buffer. 200 μL substrate was incubated with 0.2 μL UCHL3 (2.07 mg/mL) in a black 96-well plate; fluorescence monitored on Thermo Scientific Varioskan LUX (Ex 485 nm / Em 528 nm).
