Figure 2. . Basic principles of fluorescence-based homogenous assays for measuring protease activity.

Figure 2.

Basic principles of fluorescence-based homogenous assays for measuring protease activity. (a) Fluorescence intensity assay in which a fluorogenic group is linked to the carboxyl end of a peptide via an amide bond, and its fluorescence increases upon release by the action of a protease; (b) Resonance-energy-transfer-based assay in which a FRET signal can be detected when the donor and acceptor are in close proximity. The pair separates upon peptide cleavage and the FRET signal decreases; (c) Dual-label quenched-pair fluorescent assay in which the fluorescence intensity of the reporter is suppressed by the quencher because of its close proximity. The pair separates upon peptide cleavage and the fluorescence intensity from the reporter group is significantly increased; (d) Fluorescence polarization assay in which the substrate and the product give different emission polarization signals because of their different sizes (“digestive” fluorescence polarization assay) .

From: Protease Assays

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Assay Guidance Manual [Internet].
Sittampalam GS, Coussens NP, Brimacombe K, et al., editors.
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