I am having some difficulty using monoamine oxidase A. I plan on doing inhibition assays in 96-well plates, but right now I am trying to determine Km and initial velocity using kynuramine as substrate. For some reason I am not seeing any activity.
When I received MAO A, I thawed quickly at 37C and then stored 20-uL aliquots at -80C. I thaw MAO quickly and keep on wet ice. Each well of the 96-well plate contains 100 uL kynuramine (conc ranging from 50-1000 uM) at 37C. I add 100 uL of cold MAO A resulting in final MAO conc of 0.002 to 0.2 mg/mL (equal to 0.1 to 10 U/mL). I record absorbance at 360 nm for 20 min and there is no decrease in absorbance due to conversion of kynuramine. The kynuramine alone has high absorbance, so I don't believe there is anything wrong with the substrate. According to the analysis provided by supplier, there should be 285 U/mL in the 5 mg/mL MAO A as recieved. Any ideas on what is going wrong if you are familiar with this assay or MAO?
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