Black microplates are pre-coated with protease-specific antibody. The sample is added allowing both pro- and active forms of the enzyme to bind the immobilized antibody. Unbound proteases are removed following wash steps.
A protease-specific peptide substrate (green & purple) is added. The substrate features a fluorophore (green) and quencher molecule (purple) on opposite sides of the prospective cleavage site.
Active enzyme cleaves the peptide substrate between the fluorophore and the quencher molecule, increasing the distance between them. Energy from the fluorophore is now available as fluorometric signal since the quencher is no longer close enough to absorb it. The resulting signal is directly proportional to the amount of active protease bound in the initial step.
