| Assay Method Information | |
| | Lipoxygenase UV-Vis-based IC50 Assay |
| Description: | The full IC50 experiments were done with at least five different inhibitor concentrations. All reaction mixtures were 2 mL in volume and constantly stirred using a magnetic stir bar at room temperature (23° C.) with the appropriate amount of LOX isozyme [h5-LOX (˜200 nM); h12-LOX (50 nM); h15-LOX-1 (60 nM); h15-LOX-2 (200 nM)]. The protein concentrations are the total protein concentration; active protein concentration can be significantly less due to incomplete metalation. Reactions with h12-LOX were carried out in 25 mM HEPES (pH 8.0) 0.01% Triton X-100 and 10 μM AA. Reactions with the crude, ammonium sulfate precipitated h5-LOX were carried out in 25 mM HEPES (pH 7.3), 0.3 mM CaCl2), 0.1 mM EDTA, 0.2 mM ATP, 0.01% Triton X100 and 10 μM AA. Reactions with h15-LOX-1 and h15-LOX-2 were carried out in 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100 and 10 μM AA. The concentration of AA was quantitated by allowing the enzymatic reaction to proceed to completion in the presence of soybean 15-LOX-1 (s15-LOX-1). |
| Affinity data for this assay | |
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