Fluorescently Labeled Protease Substrates

Typically, fluorescence-based protease assays use peptide amidomethylcoumarin derivatives as substrates. The non-fluorescent substrate liberates a fluorescent coumarin upon proteolytic cleavage. BIOSYNTAN offers custom tailored substrates with different coumarin based fluorogenic labels.

Colored components interfere with fluorescence measurements, resulting in high background noise and reduced sensitivity. This is particularly significant with fluorophores which absorb or emit in the ultraviolet region, such as AMC.

Rhodamine based assays offer an alternative solution due to their more red-shifted excitation and emission wavelengths (AMC: Ex 380nm/Em 460nm vs. Rh110: Ex 492nm/Em 529nm). Consequently, rhodamine assays deliver fewer false positive hits in synthetic product libraries. Up to 300-fold higher sensitivity is gained when compared with AMC substrates ^[1] or AMC labeled proteins such as ubiquitin ^[2].

For more detailed information about rhodamine 110 labeled substrates, please click here.

A third approach is based on FRET systems. In this system, a protease substrate is labeled at two positions with a corresponding FRET pair. Upon proteolytic cleavage, the FRET partners are separated and the FRET effect is suppressed. Fluorescence is generated (in case of a quencher) or shifted to different wavelengths (in case of two fluorophores). This enables a positive readout with a high signal-to-noise ratio.

For more detailed information about suitable FRET systems, please click here.