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Malate Dehydrogenase Assay

Malate Dehydrogenase Assay
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Cat. #: CB020

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Malate Dehydrogenase Assay (MDH)

Cat. No. CB020

(100 Tests in 96-well plate)

Product Description

Malate dehydrogenase (MDH2) catalyzes the last reaction of the TCA or Krebs cycle, converting malate and NAD+ to oxaloacetate and NADH in the mitochondria. In reversing the reaction, MDH is also involved in gluconeogenesis, allowing oxaloacetate/malate to leave the mitochondria. Once in the cytosol, the malate is oxidized back to oxaloacetate by cytosolic MDH or MDH1, which is followed by conversion of oxaloacetate to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (PEPCK). The interconversion between malate and oxaloacetate also constitutes the essential steps of the malate-aspartate shuttle. This colorimetric assay is based on malate dehydrogenase-catalyzed oxidation of malate, where the resulting NADH can then convert a nearly colorless probe to a colored product; the intensity of the colored product is proportional to the amount of MDH in the sample, exhibiting maximum absorbance at 440nm.

Kit Components

Assay buffer, 25 mL

MDH positive control, 20 μL

Developer (10X), 0.1 mL

NAD, 0.5 mL

WST, 3.91 mg

Cofactor, 0.5 mL

Substrate, 0.5 mL

Reagents and Positive Control Preparation

1.      Diluted MDH positive control: Add 1 µl of MDH positive control into 39 µl assay buffer (8638a). Prepare diluted MDH positive control to a final volume of 10 µL/well in a 96-well flat bottom plate.

2.      Developer solution (1X): dilute developer (10X) (8638c) in assay buffer (8638a) (1:10).

3.      WST solution: reconstitute each vial of WST with 0.6 mL assay buffer (8638a). Vortex briefly and keep in the dark at -20°C until use. For longer storage, we suggest that you aliquot and store the reconstituted WST solution at -20°C, avoid repeated freeze/thaw cycles.

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