| Assay Method Information | |
| | Biological Assay |
| Description: | Reaction buffers used for the lipoxygenase assay were as follows: 25 mM HEPES (pH 7.5), with 0.01% Triton X-100. 1 mM 12-HPETE or 1 mM 15-HPETE stock solution was prepared in 25 mM HEPES with 0.01% Triton X-100.The standard curve of 12-HPETE was made by serial dilution of 0, 6.25, 12.5, 25, 50, 75 and 100 μM, and the final reaction volume in 96 Well Clear Flat Bottom UV-Transparent Microplate is 100 μL. The absorbance of the 12-HPETE at each concentration was measured at 234 nm.The standard curve of 15-HPETE was made by serial dilution of 0, 3.125, 6.25, 12.5, 25, 50 and 100 μM, and the final reaction volume in 96 Well Clear Flat Bottom UV-Transparent Microplate is 100 μL. The absorbance of the 12-HPETE at each concentration was measured at 234 nm.[0453]IC50 values were obtained by determining the % inhibition at various inhibitor concentrations. The final concentrations of the control inhibitors and test compounds in the IC50 determination assay are 0, 0.03, 0.1, 0.3, 1, 3, 10 and 20 μM. The final concentration of DMSO in the assay is 0.05%.12-LOX IC50 determination. The 12-LOX enzyme and AA were diluted in the HEPES buffer to 120 nM and 200 μM, respectively. Pre-incubation 50 μL of indicated test compounds and 25 μL 120 nM enzyme at room temperature for 5 min. The reaction is started by adding 25 μL 200 μM AA. After a short spin (1000 rpm, 15 s), incubate the reaction system for 5 min. The blank control was set by adding 25 μL HEPES to 50 μL of control inhibitor compound (MHL355) and 25 μL 200 μM AA. The final concentrations of 12-LOX enzyme and AA were 30 nM and 50 μM, respectively. The absorbance of the 12-HPETE at 234 nm was measured. |
| Affinity data for this assay | |
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