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


Catalogue Number
60-0118-050
Product Size
50 µg
Price £
£250
Accession Number
P62987
Residues Expressed
n/a
Certificate of Analysis Size
50 µg
Species
human
Source
synthetic
Quantity
50 µg
Storage
-70°C
Concentration
2mg/ml
Formulation
DMSO
Molecular Weight
9.16kDa
Stability
12 months at -70°C; aliquot as required
Protein Sequence
Accession number: P62987. C-terminally tagged with 5-TAMRA-Lys(Ub)-Gly-OH
QA; Protein Identification
Confirmed by mass spectrometry.
QA 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.