A (MAO-A) Inhibitor Screening Kit (Fluorometric)

Catalog Number MAK295 Storage Temperature –20 C

TECHNICAL BULLETIN

Product Description Monoamine oxidases (MAO, EC 1.4.3.4) are a family of Reagents and Equipment Required but Not enzymes that oxidize a wide variety of endogenous Provided. primary amines. Two isoforms, MAO-A and MAO-B,  96 well flat-bottom plate – It is recommended to use have been identified based on their substrate, inhibitor black plates specificity, and tissue localization. MAO-A can oxidize  Fluorescence multiwell plate reader primary amines such as and .  10 mM hydrogen peroxide (H2O2) MAO-A is a mitochondrial-bound enzyme that is ubiquitously expressed throughout the brain and other Precautions and Disclaimer tissues. It has been implicated in panic, , and This product is for R&D use only, not for drug, depression. household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe Several MAO-A specific inhibitors such as clorgyline, handling practices. , , , etc. have been used as , but their usage has been limited Preparation Instructions due to side effects. This MAO-A Inhibitor Screening Kit Briefly centrifuge vials before opening. Use ultrapure offers a rapid, simple, sensitive, and reliable test water for the preparation of reagents. To maintain suitable for high-throughput screening of MAO-A reagent integrity, avoid repeated freeze/thaw cycles. inhibitors. The assay is based on the fluorometric detection of hydrogen peroxide (H2O2), one of the MAO-A Assay Buffer – Bring to room temperature by-products generated during the oxidative deamination before use. Store at –20 C. of the MAO substrate (). High Sensitivity Probe – Bring to room temperature Components before use. Protect from light and moisture. Store The kit is sufficient for 100 assays in 96 well plates. at –20 C. Use within 2 months.

MAO-A Assay Buffer 25 mL MAO-A Enzyme – Reconstitute with 25 L of MAO-A Catalog Number MAK295A Assay Buffer. Mix well. Aliquot and store at –80 C.

Use within 2 months. High Sensitivity Probe (in DMSO) 0.2 mL

Catalog Number MAK295B MAO-A Substrate – Reconstitute with 110 L of water.

MAO-A Enzyme 1 vial Store at –20 C. Use within 2 months. Catalog Number MAK295C Developer – Reconstitute with 220 L of MAO-A Assay MAO-A Substrate 1 vial Buffer. Mix well. Store at –20 C. Use within Catalog Number MAK295D 2 months.

Developer 1 vial Catalog Number MAK295E

Inhibitor Control (Clorgyline) 1 vial Catalog Number MAK295F 2

Inhibitor Control (Clorgyline) – Reconstitute with 250 L Note: Always use freshly prepared MAO-A Enzyme of water to make a stock solution of 2 mM. Mix well. working solution. Don’t store the enzyme working Make a 10 M working solution by adding 5 L of solution. the 2 mM stock solution into 995 L of water. Store the stock solution at –20 C. Use within 2 months. To check the possible inhibitory effect of test inhibitors Inhibitor working solution can be stored at 4 C to on Developer, replace the 1 L of diluted MAO-A use within 24 hrs. Enzyme with 1 L of 10 mM H2O2. Mix and add 50 L to the TI well. Incubate for 10 minutes at 25 C. Storage/Stability The kit is shipped on wet ice. Storage at –20 C, MAO-A Substrate Solution Preparation protected from light, is recommended. Avoid repeated For each well, prepare 40 L of MAO-A Substrate freeze/thaw for all non-buffer components. Briefly Solution, see Table 1. centrifuge small vials prior to opening. Table 1. Procedure Preparation of MAO-A Substrate Solution Read entire protocol before performing the assay. Reagent Samples & Controls Screening Compounds, Inhibitor Control, and Blank MAO-A Assay Buffer 37 L Control Preparations MAO-A Substrate 1 L Dissolve test inhibitors into proper solvent. Dilute to 10 Developer 1 L the desired test concentration with MAO-A Assay Buffer High Sensitivity Probe 1 L before use. Add 10 L of test inhibitor (S), working solution of Inhibitor Control (IC) and MAO-A Assay Buffer (Enzyme Control; EC) into assigned wells. Mix well and add 40 L of the MAO-A Substrate Solution into each well. Mix well. Note: Preferred final solvent concentration should not be more than 2% by volume. If solvent exceeds 2% Measurement include a Solvent Control to test the effect of the Measure the fluorescence kinetically (ex = 535 nm/ solvent on enzyme activity. em = 587 nm) at 25 C for 10–30 min. Choose two points (T1 and T2) in the linear range of the plot and obtain the corresponding fluorescence values (RFU Optional: To check the possible inhibitory effect of test 1 and RFU2). inhibitors on Developer, prepare a parallel test inhibitor well (TI). Inhibitor Control-Clorgyline does not inhibit the Results Developer. Calculations Calculate the slope for all samples, including Enzyme

MAO-A Enzyme Solution Preparation Control (EC), by dividing the net RFU (RFU2 – RFU1) Dilute the Enzyme stock solution 5 times by adding values by the time T (T – T ). Calculate % Relative 2 L of MAO-A Enzyme Stock Solution into 8 L of 2 1 MAO-A Assay Buffer. For each well, prepare 50 L of Inhibition as follows: MAO-A Enzyme Solution, containing 49 L of MAO-A Assay Buffer and 1 L of Diluted MAO-A Enzyme. % Relative = (Slope of EC – Slope of S)  100 Inhibition Slope of EC Mix. Add 50 L/well into wells containing test inhibitors, Inhibitor Control, and Enzyme Control. Incubate for 10 minutes at 25 C.

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Troubleshooting Guide Problem Possible Cause Suggested Solution Cold assay buffer Assay Buffer must be at room temperature Omission of step in procedure Refer and follow Technical Bulletin precisely Assay Not Working Plate reader at incorrect wavelength Check filter settings of instrument Type of 96 well plate used It is recommended to use black plates Use the Assay Buffer provided or refer to Samples prepared in different buffer Technical Bulletin for instructions Repeat the sample homogenization, Cell/Tissue culture samples were increasing the length and extent of incompletely homogenized homogenization step. Samples with erratic Samples used after multiple freeze-thaw Aliquot and freeze samples if samples will be readings cycles used multiple times Presence of interfering substance in the If possible, dilute sample further sample Use of old or inappropriately stored Use fresh samples and store correctly until samples use Thaw all components completely and mix Improperly thawed components gently before use Use of expired kit or improperly stored Check the expiration date and store the Lower/higher reagents components appropriately readings in samples Allowing the reagents to sit for extended Prepare fresh Reaction Mix before each use and standards times on ice Refer to Technical Bulletin and verify correct Incorrect incubation times or temperatures incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly Thaw and resuspend all components before Use of partially thawed components preparing the reaction mix Pipetting errors in preparation of standards Avoid pipetting small volumes Pipetting errors in the Reaction Mix Prepare a Reaction Mix whenever possible Pipette gently against the wall of the plate Non-linear standard Air bubbles formed in well curve well Standard stock is at incorrect Refer to the standard dilution instructions in concentration the Technical Bulletin Recheck calculations after referring to Calculation errors Technical Bulletin Substituting reagents from older kits/lots Use fresh components from the same kit Samples measured at incorrect Check the equipment and filter settings wavelength Unanticipated results Samples contain interfering substances If possible, dilute sample further Sample readings above/below the linear Concentrate or dilute samples so readings range are in the linear range

SJ,MAM 04/16-1

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