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

Overview

  • Product name
    Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit
    See all Pyruvate dehydrogenase kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue Lysate, Purified mitochondria
  • Assay type
    Enzyme activity
  • Assay duration
    Multiple steps standard assay
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902) can be 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.


    The PDH complex is relatively fragile and sensitive to detergent. Please follow the sample preparation steps provided in the protocol booklet. Other preparation methods may decrease PDH activity. Our Scientific Support team is happy to answer any questions about sample prep.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Notes

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

    Store 5X Stabilizer at -20°C.

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

  • Platform
    Microplate

Properties

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

This product has been referenced in:
  • Scheffler K  et al. 8-oxoguanine DNA glycosylase (Ogg1) controls hepatic gluconeogenesis. DNA Repair (Amst) 61:56-62 (2018). Read more (PubMed: 29207315) »
  • Ravera S  et al. Concentration-dependent metabolic effects of metformin in healthy and Fanconi anemia lymphoblast cells. J Cell Physiol 233:1736-1751 (2018). Read more (PubMed: 28681917) »
See all 35 Publications for this product

Customer reviews and Q&As

1-10 of 79 Abreviews or Q&A

Answer

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 example. We agree to increase the amount of sample used. The customer’s OD450 reading for 100 ug of extracts are essentially background readings, please see the attached file. The quality of homogenate preparation is important. Protease inhibitors should be used during homogenate preparation and sample extraction. I have also inquired about any specific sample preparation tips for mouse liver tissue and will let you know when I hear back from the lab. I hope this information is already helpful to you. Thank you for your patience. Please do not hesitate to contact us if you need any more advice or information.

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Abreviews
Cells were collected, counted and resuspended in PBS to adjust the sample protein concentration to 15 mg/mL. Then, samples were solubilized with Detergent (9:1), incubated on ice for 10 min and centrifuged at 1,000 g for 10 min at 4 ºC. The supernatants were diluted in Assay Buffer to the appropriated concentration within the linear working range for the assay, loaded to each well of the microplate and incubated at room temperature for 3 h. For the measurement, wells were emptied and 200 μL of Assay Solution were added. The absorbance
was measured at 450 nm using a kinetic program with readings every 25 sec for 30 min. PDH activity was normalized by protein content in the supernatant, determined by the bicinchoninic acid (BCA) assay.
It is important to notice that absorbance rapidly increases with time and therefore the time spent when adding the Assay Solution between wells is crucial.

Abcam user community

Verified customer

Submitted Oct 03 2018

Abreviews
The product was tested on mitochondria isolated from fetal sheep hearts. Activity was obtained with sheep heart mitochondria and the raw data ( starting from 12.5 ng/well to 400ng/well) is attached below.

Abcam user community

Verified customer

Submitted Jan 09 2018

Answer


It is not necessary to avoid EDTA in the protease inhibitor cocktail. EDTA should not affect the pyruvate dehydrogenase enzyme in the sample or the functioning of the assay.

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Answer

Thank you for your reply.

The laboratory have advised that probably the best in this situation would be to maintain the DCA in all the cell washes after treatment and during the assay itself without NaF, so that full activation of PDH can be observed. The effect of DCA in-vitro is reversible as soon as DCA is removed from the system.

Sodium fluoride is useful to measure the endogenous activity of PDH to ensure that any level of inhibition by the kinases is not counteracted by phosphatases during sample preparation.


I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

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Answer


I contacted the laboratories who have confirmed that for phosphatase inhibition we recommend 10mM NaF for use with this kit. A 500mM stock solution can be prepared in water and stored at -20C.

ab65621 is an appropriate protease inhibitor cocktail.
https://www.abcam.com/index.html?datasheet=65621 (or use the following: https://www.abcam.com/index.html?datasheet=65621).

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Answer

Please see below for further clarification regarding the following questions:

Is it OK to extract mitos from frozen tissues?

A: yes, you can extract mitos from frozen tissues but be aware that freezen/ thaw cycle would weaken the cell membrane and cause less recovery of intact mitochondria.
Are there differences between the extraction from frozen mitos versus fresh mitos?

A: yes, there will be slightly loss if you extract enzyme from frozen mitos versus fresh mitos due to the degradation. Adding protease inhibitor to your mito prep would minimize the loss.
Does the extraction time point from a frozen tissue matters?

A: No, as long as the tissues are properly frozen, the variant between different time points should be minimum (within weeks). However, multiple freezen/thaw cycle will cause difference. If you plan to extract sample from different days, make sure you make aliquots of your samples. if you plan to compare the enzyme activity from different samples, we highly recommend to prep the samples under the same condition.

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Answer

Thank you for contacting us.



Nome of the reagents in this kit contain thymine.





I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.



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Answer

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 questions, including the amount of modification enzyme, will be clear after reading the protocol below:
http://www.mitosciences.com/PDF/endogenous_pdh_activity_phosphorylated_dephosphorylated_states.pdf
2. To set up appropriate extraction conditions, optimized ratio of total protein to kit’s Detergent is required. We usually perform total protein determination by BCA assay on 0.25% SDS lysate of the sample. Please note to do the SDS extraction on a separate aliquot of the sample that will not be used further in the PDH activity assay
3. The dilution of the extract with 1X Buffer is important part of the sample preparation, the properly prepared extract is too concentrated to be measured without dilution. 4. No need of incubation, this is simply a washing procedure in the presence of a stabilizer.

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1-10 of 79 Abreviews or Q&A

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