What is the detection principle? In a protease activity assay, how can I determine whether the background fluorescence of the Gly-Pro-AMC substrate is significant?
Binbin Lee,
Gly-Pro-AMC has background fluorescence, but it is generally low.
Gly-Pro-AMC is a fluorogenic substrate which is cleaved by enzymes in protease assays, particularly the peptide bonds after proline residues. When the substrate is cleaved by a protease, the AMC moiety is released, which is highly fluorescent.This released AMC contributes to the overall fluorescence signal, which is what is measured in the assay to determine protease activity.
To accurately quantify protease activity, you may subtract the background fluorescence from the total fluorescence signal. This can be done by measuring the fluorescence of the substrate solution without any protease present (blank), or you may use a protease inhibitor to block protease activity and measure the remaining fluorescence (inhibitor control). Then subtract the blank or inhibitor control value from the experimental readings.
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