Table 112-hLO qHTS assay protocol for 1536-well plate format

StepParameterValueDescription
1Reagent3 μLEnzyme: 120 nM 12-hLO final conc.
2Compound23 nLCompound library
3Control23 nLIntraplate titration
4Incubation time15 minCompound interaction with target
5Reagent1 μLSubstrate: AA 40 μM final conc.
6Incubation time5 minEnzymatic reaction
7Reagent4 μLStop solution
8Time, speed15 sec, 1000 rpmCentrifugation
9Incubation time30 minutesChromogenic detection development
10Assay readout405 and 573 nmViewLux absorbance read
StepNotes
1Black clear-bottom plates, single tip dispense, enzyme in columns 1, 2, 5–48, vehicle in column 3 and 4.
2Pintool transfer of compounds to assay plate columns 5–48. 57.5 μM to 0.7 nM titration series.
3Pintool transfer of control inhibitor to column 2. Three-fold, 16 pt dilution in duplicate.
4Room temperature incubation in auxiliary hotel.
54-tip dispense of arachidonic acid (AA), 1536 wells.
6Room temperature incubation in auxiliary hotel.
74-tip dispense of divalent iron/xylenol orange in dilute sulfuric acid, 1536 wells.
8Plate centrifugation to remove bubbles.
9Room temperature incubation in auxiliary hotel.
10Endpoint measurement of reaction progress.

From: Selective Small Molecule Inhibitors of 12-Human Lipoxygenase (12-hLO)

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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