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

Reviews (4)Q&A (76)References (84)
Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
  • Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
  • Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
  • Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

Key features and details

  • Assay type: Enzyme activity
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 3 hr 30 min
  • Sample type: Cell culture extracts, Purified mitochondria, Tissue Lysate

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 time

    3h 30m

  • 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 reader

Images

  • Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    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.
  • Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    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).
  • Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    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).
  • Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    Functional Studies - Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)
    Principle of Pyruvate dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit (ab109902)

References (84)

Publishing research using ab109902? Please let us know so that we can cite the reference in this datasheet.

ab109902 has been referenced in 84 publications.

  • Godsman N  et al. Metabolic alterations in a rat model of takotsubo syndrome. Cardiovasc Res 118:1932-1946 (2022). PubMed: 33711093
  • Paruchuru LB  et al. The Critical Role Played by Mitochondrial MITF Serine 73 Phosphorylation in Immunologically Activated Mast Cells. Cells 11:N/A (2022). PubMed: 35159398
  • Xia H  et al. Insulin action and resistance are dependent on a GSK3ß-FBXW7-ERRa transcriptional axis. Nat Commun 13:2105 (2022). PubMed: 35440636
  • Kay EJ  et al. Cancer-associated fibroblasts require proline synthesis by PYCR1 for the deposition of pro-tumorigenic extracellular matrix. Nat Metab 4:693-710 (2022). PubMed: 35760868
  • Visavadiya NP  et al. Distinct glycolytic pathway regulation in liver, tumour and skeletal muscle of mice with cancer cachexia. Cell Biochem Funct 39:802-812 (2021). PubMed: 34129243
View all Publications for this product

Customer reviews and Q&As

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1-10 of 80 Abreviews or Q&A

Did not seem to work for sheep

 Below average Excellent 5/5 (Ease of Use)
Abreviews
abreview image
Unfortunately, unlike the item AB-ab185440 which worked fine for sheep aKGDH, item AB-ab109902 did not show specific activity against sheep PDH.

About 1g liver tissue was harvested and washed throughly with ice cold PBS buffer. Resuspend the tissue in 5mL of ice cold PBS and homozinised by using a rotor homogenizer. homozinised component was subjected to centrifugation for 10 min at 4°C at 5000g. Collected supernatant was transferred to new tubes then re centrefused at 20000g for 10min at 4°C. The obtained clear lysate was used to measure the protein concentration by Coomassie brilliant blue G method and quantification was determined based on intensity of blue stains at 595nm (protein quantification kit-rapid, Sigma-Aldrich, USA). For the PDH assay reaction a total of 100ug protein was loaded to each well. Then, followed reaction steps specified in protocol (Abcam, Cambridge UK). The PDH actvity is expressed as the intial rate of reaction, determined from the slope of the curve generated at 450nm. The increased rate of enzymatic reaction was monitored at absorbance of 450nm over the time.
The rate of reaction was caluculated at between two time points for all the samples where the absorbance was considered as linear.
Rate (mOD/min)= Abs2-Abs1
Time (min)

Results: The slight increased absorbace depends on time show the rate enzymatic recation in samples (Fig 1). The Fig 2 shows the relationship between PDH signaland sample load (100ug/well) over time.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Verified customer

Submitted Mar 04 2021

Quantitative PDH enzyme activity determination

 Good Average 3/5 (Ease of Use)
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Submitted Oct 03 2018

ab109902 Pyruvate Dehydrogenase (PDH) Enzyme Activity Microplate Assay Kit

 Average Average 3/5 (Ease of Use)
Abreviews
abreview image
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.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

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Verified customer

Submitted Jan 09 2018

Question

uses mouse liver homogenates activity is low used 100 ug extract per well, conc = 30 mg/ml readout OD 450 nm is between 2 and 3, but blank is 1.8 hepG2 cell data show high signal for 500 ug/well Has liver tissue been tested? How much more sample should be used for a good signal?

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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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Question

I have a query regarding your ab109902 Pyruvate Dehydrogenase (PDH) Enzyme activity microplate assay kit. 

In your protocol you recommend using an EDTA-free Protease inhibitor cocktail. I would like to enquire whether it is absolutely necessary that the inhibitor is EDTA free, and if so, why this is the case. 


 

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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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Question

I have searched through some literature and I have found that the dichloroacetate (DCA) is also used for inhibition of kinase activity. Could you confirm that DCA can be used together with NaF in a single lysis mixture, please? Did you study the concentration of DCA required for your assay as well?


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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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Question

Inquiry: Dear Sir or Madam, Could you offer some guidance about the lysis buffer components, please? It is mentioned in the ab109902 booklet, that it may be necessary to include PDH phosphatase and kinase inhibitors in the sample preparation step (see page 5). However, such components are not included in the ab109902 kit. Could you share some knowledge, what kind of inhibitors can be used with the kit to preserve the endogenous phosphorylation status and also not interfere with the reading step (enzymatic reaction)? We are receiving results that suggest elevated PDH activity in the 3T3-L1 cells. We would like to see, if it resulted from constitutively activated PDH or exogenous dephosphorylation of PDH during sample preparation.

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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/Protease-Inhibitor-Cocktail-ab65621.html (or use the following: https://www.abcam.com/Protease-Inhibitor-Cocktail-ab65621.html).

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Question

I am removing brains from a number of rats. I'd like to be able to remove all the brains in one go and store them until I'm ready to process the tissue all at the same time. I'd like to use the tissue, once processed using ab110169, for the following kits 1) ab109721, 2) ab110413 and ab109902.
So the main question is, can I store the brains by freezing (eg. liquid nitrogen to -80 deg C) and thaw the brains and produce extracts that will produce viable sample for use with all 3 kits noted above.

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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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Question

I am removing brains from a number of rats. I'd like to be able to remove all the brains in one go and store them until I'm ready to process the tissue all at the same time. I'd like to use the tissue, once processed using ab110169, for the following kits 1) ab109721, 2) ab110413 and ab109902.
So the main question is, can I store the brains by freezing and then thaw the brains and produce extracts that will produce viable sample for use with all 3 kits noted above. Thanks!

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Answer

Yes, you can use frozen tissues as sample for all three kits. As a reminder, please follow each individual protocol included with each kit to prepare samples for that particular assay.

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Question

I'd like to know if any of the reagents in this kit contain any form of thymine, as I want my cells to be thymine deficient during the experiment.
If any of the reagents do contain thymine, can I please be provided the recipe to make this reagent so I can make it without thymine? Alternatively I would be able to purchase this buffer separately if its available thymine free.

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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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1-10 of 80 Abreviews or Q&A

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