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.
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