From 2011.igem.org

Revision as of 01:44, 23 September 2011

Enzyme Assay

Substrate

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:
      • Cutoff: