MitoSciences (ab109902)

Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

Overview

  • Product namePyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay KitSee all Pyruvate dehydrogenase kits ...
  • Detection methodColorimetric
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts, Tissue
  • Assay typeEnzyme activity
  • Assay durationMultiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    This rapid activity microplate kit is used to determine the activity of PDH in a human, bovine, mouse, or rat sample. The PDH enzyme is immunocaptured within the wells of the microplate and activity is determined by following the reduction of NAD+ to NADH, coupled to the reduction of a reporter dye to yield a colored reaction product with an increase in absorbance at 450 nm at room temperature. Included for performance of the activity assay are buffer, detergent, reagent mix, and a 96-well microplate with monoclonal antibody pre-bound to the wells of the plate, allowing for a stream-lined assay.

    This assay is optimized for use with whole tissue extract when the amount of total material available for assay is 20-100 µg or more. If using cell extract of cultured cells 1 mg of material is required due to the very low levels of enzyme and reduced levels of mitochondria in the extract.

  • Notes

    Store 20X Buffer, Detergent, and Microplate at 4°C.

    Store 5X Stabilizer and 100X Reagent Dye at -20°C.

    Store 20X Reagent Mix and 100X Coupler at -80°C.

  • Tested applicationsFunctional Studies more details
  • PlatformMicroplate

Properties

  • Alternative names
    • PDC
    • PDH
    • PDHC

Applications

Our Abpromise guarantee covers the use of ab109902 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Functional Studies Use at an assay dependent dilution.

Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit images

  • Mitochondria, tissue extracts and whole cultured cell extracts all show linear relationships between signal and sample load at limiting concentrations. The rates shown were determined as change in OD over time, and these are best represented as change in milliOD per minute.
  • Example of microplate reader recorded data from bovine heart mitochondria (100 µg/well) (top trace) and 2-fold dilutions (stepwise lower traces) using Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902).
  • Schematic diagram showing the reaction involved in Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902).
  • Principle of Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

Protocols

References for Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

This product has been referenced in:
  • Morten KJ  et al. Differential regulation of HIF-mediated pathways increases mitochondrial metabolism and ATP production in hypoxic osteoclasts. J Pathol 229:755-64 (2013). Read more (PubMed: 23303559) »
  • Phillips BE  et al. Effect of colon cancer and surgical resection on skeletal muscle mitochondrial enzyme activity in colon cancer patients: a pilot study. J Cachexia Sarcopenia Muscle 4:71-7 (2013). Functional Studies ; Human . Read more (PubMed: 22648738) »

See all 12 Publications for this product

Product Wall

Thank you for contacting us. I have heard back from the lab: Yes, liver tissues were tested with ab109902; for example extract of mouse liver homogenate. For extract of liver homogenate, 100 – 500 ug/200 uL should be used, please see attached ex...

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Please find the answers to your questions from my colleagues at Miosciences, who are designing this kits:
1. The dephosphorylation modifications can be done after the immunocapture of the PDH enzyme to the plate. Many related concerns and question...

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Product ab110338 contains whole mitochondria in iso-osmotic buffer. There is no need to measure protein concentration of this product; the product is supplied at 5.5 mg/mL. Simply adjust the protein concentration with PBS to 5.3 mg/mL and add dete...

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There is no need to homogenize mitochondrial preparation in PBS.

I'd recommend measuring protein concentration of mitochondrial PBS suspension on an aliquot diluted in a detergent (for example 100x dilution of mitochondria suspension in 0.25%...

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It is recommended to prepare tissue homogenate in PBS at 23.7 mg/mL. One can use as a guideline the first part of ab110168 protocol. Just substitute all buffers with PBS. In general what you need to do is just mince/grind/homogenize tissue in PBS witho...

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Pyruvate dehydrogenase activity in HCT116 cells

Average Average 3/5 (Ease of Use)
Abreviews
We wanted to analyse the enzymatic activity of pyruvate dehydrogenase (PDH) in human HCT116 cells dependent on the glucose level within the media. Therefore we cultivated these cells in DMEM with high (4.5 g/l) and low (1 g/l) glucose for 48 h. They were harvested and lysed in PBS. We used whole cell lysates to purify proteins. The PDH activity was spectroscopically measured by a change in absorbance at 450 nm. The absorbance was measured every minute for 30 min and kinetics were analysed with GraFit. The changed absorbance per minute was plotted against protein mass used to load the plate (fig. A and B). The blank value was calculated from four blanks in each test.
Unfortunately we only obtained significant activities at 500 µg protein or higher (fig. A). Thus we repeated the test with higher protein concentrations (fig. B). But in both test, no substantial difference between cells cultivated in high or low glucose was observed.
For these assays we had the problem that the four blanks in each test had a high inter-plate variation. This may be due to our plate reader or to the plate delivered with the assay kit. Therefore you should always use technical replicates. Additionally we think that problems can easily appear if the protein quantification during the test is incorrect and accordingly the loading of the plate.
In general the assay kit is not easy to use but gives a quick method to analyse the PDH activity.
Username

Mr. Christian Marx

Verified customer

Submitted Oct 04 2013

Freezing will not be an issue. I have not seen data comparing fresh and frozen samples but if you freeze all of your samples the same way, ideally flash-freezing in liquid nitrogen before storage at -20C, you should be able to obtain good data. The dat...

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The kit should yield the same results whether the tissue is fresh or frozen, as long as the original tissue was frozen quickly after disection and quick-flash frozen in liquid nitrogen. In fact, the graphs in the kit protocol of mouse and rat heart hom...

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The T-PER buffer provided by Thermo scientific seems compatible with the kit ab109902 as this does not contain any detergent or SDS. You can try this buffer.
Please note that we have not tested this buffer with this kit so if you get unrealistic re...

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The ∆OD can be converted to ∆milliOD simply multiply ∆OD by a thousand. In order to calculate the rate, this value will need to be divided by the number of minutes.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"