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NameCatalogue NumberSizePriceAdd to Basket
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Catalogue Number:60-0118-050Size:50 µgPrice:£250Add To Basket
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Specieshuman
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Sourcesynthetic
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Quantity50 µg
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Storage-70°C
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Concentration2mg/ml
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FormulationDMSO
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Molecular Weight9.16kDa
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Stability12 months at -70°C; aliquot as required
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Protein SequenceAccession number: P62987. C-terminally tagged with 5-TAMRA-Lys(Ub)-Gly-OH
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QA; Protein IdentificationConfirmed by mass spectrometry.
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QA; ActivityActivity Assay: The activity of 5-TAMRA-Lys(Ub)-Gly-OH was validated by determining the decrease in mP (measured at Excitation 540, Emission 590) resulting from cleavage of the fluorophore (TAMRA) from Ubiquitin after incubation with UCHL3 (deubiquitylase). UCHL3 was incubated with 5-TAMRA-Lys(Ub)-Gly-OH and fluorescence intensities were measured in the S (parallel) and P (perpendicular) at four time points (0min, 30min, 60min and 90min), from this data mP values were calculated.
A shift in both the excitation and the emission toward longer wavelength helps overcome problems of compound autofluorescence in screening assays. One such substrate is the rhodamine based ubiquitin-eN-(aN-tetramethyl-rhodamine)lysine (Ubiquitin-Lys-TAMRA), which mimics the naturally occurring isopeptide bond Between the C-terminus of ubiquitin and the e-amino group of a lysine residue of an ubiquitinated protein (Tirat et al., 2005). Cleavage of the isopeptide bond results in a decrease of fluorescence polarization, which makes Ubiquitin-Lys-TAMRA suitable for high-throughput screening applications (Hassiepen et al., 2007). Fluorescence polarization, in contrast to fluorescence intensity, allows a ratiometric read-out of the activity and is thus less sensitive to autofluorescing or quenching caused by test compounds (Tirat et al.,2005).