Assay Method Information

Assay Name:  Inhibition Assay
Description:  A stock solution was prepared using a human MAO-B enzyme (purchased from Aldrich) and a Amplex Red monoamine oxidase assay kit according to a preparation manual. The kit includes a 5x reaction buffer, an Amplex red reagent (1 mg), HRP, DMSO, H2O2, p-tyramine (substrate of MAO-A, B), benzylamine (substrate of MAO-B), clorgiline (inhibitor of MAO-A), and pargyline (inhibitor of MAO-B). Among these reagents in the kit, benzylamine was used as a substrate for MAO-B, and pargyline was used as an MAO-B inhibitor. A solution as overall substrates was prepared as follows. 200 ul of a solution of 1 mg of Amplex red sufficiently dissolved in 200 ul of DMSO, 100 ul of a mixed solution of HRP and 1 ml of a 1x buffer, 200 ul of a solution of benzylamine dissolved in 1.2 ml of dH2O were added to 9.5 ml of a 1x buffer to reach a total volume of 10 mL, which is sufficient for 100 wells. 0.5 ul of a mixture of MAO-B inhibitor pargyline and 1 ml of dH2O was put into each well. First, the activity of MAO-B was determined using 10 uM of the synthesized compound.  96 wells were injected with positive and negative types, and the wile type. The positive type included only substrate and hydrogen peroxide, and the negative type included only substrate. For the wild type, corresponding wells were injected with the enzyme, substrate, and MAO-B inhibitor, but with no synthesized compound. Afterward, 2 ul of the synthesized compound (1 mM) was added into each well, and the human MAO-B enzyme was put only into the 1st row of wells. 0.5 ug of the human MAO-B was put into each well along with 100 ul of a 1x buffer. The human MAO-B enzyme was put into the 2nd row of the wells along with 0.5 ul of a pargyline, the MAO-B inhibitor. To reduce an experimental error for accuracy, the test was repeated three times for each compound. After 30 minutes, 100 ul of the substrate solution was added into each well in a darkroom. The test was performed in the darkroom due to light sensitivity of the Amplex reagent. Finally, a total volume of the reaction solution per well reached 200 ul. After about 2 to 3 hours, chromophoric degrees of the samples were measured. A variation in data values for the 1st and 2nd rows of the wells indicates the pure reaction activity of the MAO-B enzyme with the substrate. Using the samples with the synthesized compound the remaining activity of MAO-B after inhibited by the synthesized compound may be determined. This is because the activities of the other enzymes excluding the MAO-B enzyme may be excluded through this method. Compounds with high inhibitory activity at a concentration of 10 uM were screened from among the synthesized compounds at a compound concentration of 10 uM. Afterward, concentration-dependent IC50 values of these compounds may be obtained through an activity assay at different concentrations of 0.001 uM, 0.01 uM, 0.1 uM, 1 uM, and 10 uM.
Affinity data for this assay

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