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Pyruvate dehydrogenase Assay (PDH)

Pyruvate dehydrogenase Assay (PDH)
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Cat. #: CB021

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Pyruvate dehydrogenase Assay (PDH)

Cat. No. CB021

(100 Tests in 96-well plate)

Product Description

Pyruvate dehydrogenase (PDH) is the first component enzyme of the pyruvate dehydrogenase complex (PDC). As a key regulator of tricarboxylic acid (TCA) cycle flux, the PDC catalyzes the conversion of pyruvate into acetyl-CoA and regulates the entry of glucose-carbons into the TCA cycle. PDC deficiency is one of the most common neurodegenerative disorders associated with abnormal mitochondrial metabolism. This colorimetric assay is based on pyruvate dehydrogenase-catalyzed oxidation of pyruvate, 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 PDH in the sample, exhibiting maximum absorbance at 440nm.

Kit Components

·       Assay buffer, 25 mL

·       PDH positive control, 20 μL

·       Developer (10X), 0.1 mL

·       NAD, 2.0 mL

·       WST, 3.91 mg

·       Substrate, 1.6 mL

·       Cofactor, 1.7 mL

Reagents and Positive Control Preparation

1.      Diluted PDH positive control: Add 1 µl of PDH positive control into 9 µl assay buffer. Prepare diluted PDH positive control to a final volume of 10 µL/well in a 96-well flat bottom plate.

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

3.      WST solution: reconstitute each vial of WST with 0.6 mL assay buffer. 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.

Procedure (96-well plate)

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