Team:Washington/Protocols/Purified Enzyme Assay


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Purified Enzyme Assay


The substrate was ordered at a 20% discount from Anasepc in support of Undergraduate education. The peptide Motif PQPQLP commonly found in gluten was synthesized with a fluorophore (5-FAM) or Quencher (QXL52) available from anaspec. It is denoted as the following

Ordered Peptide (95% Purity): QXL520-PQPQLP-K(5-FAM)-NH2

  • Assay
    • Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
    • Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)
      • Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
    • Monitor the reaction with the SpectraMax
      • SpectraMax Settings:
        • Excitation:
        • Emission:
        • Sensitivity Level:

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