Team:Washington/Protocols/Purified Enzyme Assay

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=Enzyme Assay=
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
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==Substrate==
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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
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Ordered Peptide (95% Purity):  QXL520-PQPQLP-K(5-FAM)-NH2
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*'''Assay'''
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**Add 90uL of 5uM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
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**Start reaction by adding 0.0125mg/mL enzyme (try to avoid bubbles and pippette quickly, but accurately)
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***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
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**Monitor the reaction with the SpectraMax
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***SpectraMax Settings:
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****Excitation:
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****Emission:
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****Cutoff:
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''
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&nbsp; &nbsp; &nbsp;
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</div>

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:



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