Enzyme substrates based on FRET (fluorescence resonance energy transfer) are extremely sensitive tools for determining protease activity, especially when there is a requirement for specific amino acids on both sides of the cleavage site in the target protein or peptide. A fluorescent group and a quencher group are attached to either end of the peptide sequence containing the cleavage site. The peptide should be just long enough to contain the minimum sequence requirements of the protease. In the intact molecule, fluorescence resonance energy transfer between the quencher and the fluorophore leads to low fluorescence. On cleavage of the peptide by the protease, the quencher and fluorophore are separated giving a large increase in fluorescence. To maximize the FRET effect, the absorbance spectrum of the quencher and the emission spectrum of the fluorophore should have similar maxima.
CRB has a wealth of experience in the synthesis of FRET peptides and is always happy to advise on peptide design and help with the selection of the most appropriate dye and quencher for your application.
CRB offers dyes compatible with all commonly used excitation sources. The most frequently requested can be found using our on-line dye selector, together with appropriate quencher for FRET applications. If you require a peptide labelled with a dye not listed here, please enquire as we have access to many more.