Pyruvate dehydrogenase (PDH) Enzyme Activity Dipstick Assay Kit
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(29 Publications)
- FuncS
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Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Dipstick Assay Kit (AB109882)
An example using the ab109882 to measure PDH activity in human fibroblast sample. Based on the standard curve, 160 µg of protein extract were loaded onto a dipstick for each sample. The figure shows two developed dipsticks, a control sample and a sample from a patient with a mutation in the E1-alpha subunit of PDH. The analysis of the signal intensity and interpolation from the standard curve showed that the patient sample had 8% the PDH activity of the control.
- FuncS
Supplier Data
Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Dipstick Assay Kit (AB109882)
An example using the ab109882 to measure PDH activity in human fibroblast sample.
Developed dipsticks from a 1 : 2 dilution series using a positive control sample and the associated standard curve. Starting material was 160 µg of fibroblast protein extract in a 100 µL reaction volume (50 µL sample in Sample buffer plus 50 µL of Blocking solution). The dipsticks were developed for 1 hour in Activity buffer.
- FuncS
Supplier Data
Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Dipstick Assay Kit (AB109882)
Abcam's enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. Dipstick ELISA Kits extend this concept by utilizing the well-established lateral flow concept, wherein capture antibodies are striped onto nitrocellulose membrane and a wicking pad draws the sample through the antibody bands. All of our ELISA kits utilize highly validated monoclonal antibodies and proprietary buffers, which are able to capture even very large enzyme complexes in their fully-intact, functionally-active states.
Reactivity data
Product details
ab109882 is used to quantify the activity of the PDH enzyme complex from human, bovine, mouse, rat and pig samples. This is accomplished by immunocapturing PDH with an anti-PDH antibody immobilized on a defined section of a dipstick. The enzyme complex is immunocaptured in its native form and activity is measured by the reaction scheme. PDH activity is visualized by coupling PDH-dependent production of NADH to the reduction of NBT in the presence of excess diaphorase, forming an insoluble intensely colored precipitate at the capture line. The signal intensity is measured by a dipstick reader or analyzed by other imaging systems such as a flatbed scanner.
Purified mitochondria can be used in this assay. However, homogenized tissue and whole cells can also be used without the need for mitochondrial isolation.
Pyruvate dehydrogenase activity is regulated by PDH kinase and PDH phosphatase. Cells grown in glucose media derive most of their energy from glycolysis; therefore, most of their PDH complex may be in an inactive phosphorylated form at the time of isolation. Growing cells in alternate carbon sources such as glutamine/galactose may up-regulate PDH activity. This kit does not include PDH kinases, PDH phosphatases or their respective inhibitors. These may be incorporated into the assay at the user's discretion.
Store dipsticks at room temperature out of direct sunlight in their provided desiccant container. Avoid high humidity conditions.
Store 20X Reagent Mix, Detergent, and Diaphorase at -80°C, but these may be aliquoted upon receipt to prevent freeze/thaw cycles.
Store NBT in the dark at -80°C.
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Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
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Biological function summary
The actions of pyruvate dehydrogenase serve as a bridge between glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate derived from glucose is transformed into acetyl-CoA before entering the TCA cycle for further energy extraction. The PDH complex ensures efficient energy production by tightly regulating the flow of carbon into the TCA cycle. Regulation occurs through phosphorylation by specific PDH kinases which inactivate E1. This mechanism integrates signals from energy status and substrates availability modulating the carbohydrate metabolism.
Pathways
Pyruvate dehydrogenase is a central player in cellular respiration and energy metabolism. It connects glycolytic pathways with the TCA cycle facilitating energy conversion in eukaryotic cells. Key related proteins involve pyruvate kinase (which generates pyruvate) and citrate synthase (which uses acetyl-CoA) ensuring synchronized activity between upstream and downstream metabolic processes. The proper function of PDH activity is necessary for maintaining the metabolic flow with the PDH complex serving a gating role in the energy pathways.
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Publications (29)
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Orphanet journal of rare diseases 19:442 PubMed39609877
2024
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Orphanet journal of rare diseases 18:80 PubMed37046296
2023
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International journal of molecular sciences 23: PubMed36499021
2022
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Orphanet journal of rare diseases 17:311 PubMed35945593
2022
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Orphanet journal of rare diseases 16:201 PubMed33952316
2021
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Cell reports 33:108333 PubMed33147467
2020
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Proceedings of the National Academy of Sciences of 116:25389-25391 PubMed31792175
2019
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Frontiers in cardiovascular medicine 5:195 PubMed30740395
2019
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JCI insight 4: PubMed30626749
2019
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International journal of molecular sciences 19: PubMed30213070
2018
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