Ubiquitin-Lys-TAMRA (5-TAMRA-Lys(Ub)-Gly-OH)

Ubiquigent

Catalog No: UBI-60-0118-050

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Species Reactivity: human 
Source: synthetic  
Formula: DMSO  
Concentration: 2mg/ml  
Molecular Weight: 9.16kDa  
Protein Sequence: Accession number: P62987. C-terminally tagged with 5-TAMRA-Lys(Ub)-Gly-OH  
Quality Control Protein Identification: Confirmed by mass spectrometry.  
Quality Control Activity: Activity 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.  

Background

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).

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.
Tirat A, Schilb A, Riou V, Leder L, Gerhartz B, Zimmermann J, 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.