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Team:Washington/Protocols/Purified Enzyme Assay
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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 | 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 | ||
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Latest revision as of 23:16, 28 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:
- SpectraMax Settings:
