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Amino Acid Sequence: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
Applications: Useful for assaying ubiquitin C-terminal hydrolytic activity, for studying the activity and specificity of ubiquitin and ubiquitin-like hydrolases, and for screening for modulators of deubiquitylase activity.
Assay Conditions: Release of rhodamine fluorescence by UCH enzymes can be monitored using Ex485 nm and Em535 nm wavelengths, respectively.
Background: While the disubstituted rhodamine moiety in Ub-Rho110-G is essentially non-fluorescent, cleavage results in a mono-substituted rhodamine, Rho110-G, which exhibits intense fluorescence when excited at 485 nm. The longer excitation and emission wavelengths (Ex485 nm, Em535 nm) of the rhodamine fluorophore reduces the risk of artifacts in screens due to autofluorescence, which can make ubiquitin-rhodamine more appropriate than Ubiquitin-AMC for some compound screening and profiling assays.
Biological Activity: Rhodamine-N-term-ubiquitin gives a strong signal in the range of 0.1-1 ?M, depending on exact experimental conditions.
Description: Ubiquitin-rhodamine 110 is a quenched, fluorescent substrate for deubiquitylases, especially ubiquitin C-terminal hydrolases. Cleavage of the amide bond between the C-terminal glycine of ubiquitin and rhodamine results in an increase in rhodamine fluorescence at 535 nm (Exc. 485 nm).
Format: Lyophilized solid
Genbank: P62987 (ubiquitin)
Purity: ≥95% by RP-HPLC.
Solubility: Soluble in DMSO (10 mg/ml) or aqueous buffers
Storage Stability: Store in the dark at or below -80°C. Stable as supplied for up to 1 year when stored dessicated at -80°C. Store DMSO solutions at -80°C for up to 1 month. Avoid freeze/thaw cycles of solutions.
Tags:
Uniprot: P09936
Warnings: Protect from light.
Biosafety Level: Not applicable (BSL-1)
References: 1. Tirat, A., et al. 2005. Synthesis and characterization of fluorescent ubiquitin derivatives as highly sensitive substrates for the deubiquitinating enzymes UCH-L3 and USP-2. Anal. Biochem. 343, 244-255.
2. Hassiepin, U., et al. 2007. A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine 110-glycine as substrate. Anal. Biochem. 371, 201-207.
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