QuantiChrom™ Pyruvate Dehydrogenase Assay Kit

Protocol SDS

Application

• For assessing PDH activity in biological samples and screening and evaluation of PDH modulators

Key Features

• High sensitivity and wide linear range. The detection limit is 0.87 U/L, with linearity up to at least 278 U/L PDH in a 96-well plate assay.
• Homogeneous and simple procedure. Simple “mix-and-measure” procedure allows reliable quantitation of PDH activity within 20 minutes.
• Robust and amenable to HTS. All reagents are compatible with high-throughput liquid handling instruments.

Method

• OD565nm

Samples

• Biological samples

Species

• All

Procedures

• 20 min

Size

• 100 tests in 96-well plate

Detection Limit

• 0.87 U/L

Shelf Life

• 6 months

More Details

PYRUVATE DEHYDROGENASE (PDH) (E.C. 1.2.4.1) is an oxidoreductase which catalyzes the decarboxylation of pyruvate to form acetylated dihydrolipoamide in a thiamine pyrophosphate (TPP)-dependent reaction. PDH is the E1 component of the PDH complex that ultimately produces acetyl-CoA. The PDH complex connects Glycolysis to the Citric Acid Cycle. This non-radioactive colorimetric PDH assay is based on the reduction of the tetrazolium salt MTT in a Phenazine Methosulfate (PMS) coupled reaction to a reduced form of MTT which exhibits an absorption maximum at 565 nm. The intensity of the purple color formed is directly proportional to the PDH activity.

Does the kit include Pyruvate Dehydrogenase enzyme?

No, the researcher must provide their own Pyruvate Dehydrogenase enzyme, but the kit was developed using purified PDH from porcine heart muscle (Sigma P-7032).

Does this kit measure the PDH complex activity?

This kit is used to measure the enzyme activity of the E1 subunit of the PDH complex, so neither NAD nor FAD are required for the assay.

Can I let the plate sit out between the T0 minute read and the T20 minute read?

The plate needs to be protected from the light during its entire incubation as the developing reagent (PMS-MTT) is very photosensitive.

For more detailed product information and questions, please feel free to email or call us at 1-510-782-9988 x 2. For more general information regarding our assays, please refer to Technical Support.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.