Overview

  • Product name

    Pyruvate dehydrogenase (PDH) Enzyme Activity Dipstick Assay Kit
    See all Pyruvate dehydrogenase kits
  • Sample type

    Cell culture extracts, Tissue
  • Assay type

    Sandwich (quantitative)
  • Species reactivity

    Reacts with: Mouse, Rat, Cow, Human, Pig
  • Product overview

    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.

  • Notes

    Store dipsticks at room temperature out of direct sunlight in their provided desiccant container. Avoid high humidity conditions.

    Store Sample buffer at 4°C.

    Store 20X Reagent Mix, Detergent, and Diaphorase at -80°C, but these may be aliquoted upon receipt to prevent freeze/thaw cycles.

    Store Blocking solution at -20°C.

    Store NBT in the dark at -80°C.

  • Tested applications

    Suitable for: Functional Studiesmore details
  • Platform

    Reagents

Properties

Applications

Our Abpromise guarantee covers the use of ab109882 in the following tested applications.

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

Application Abreviews Notes
Functional Studies Use at an assay dependent dilution.

Images

  • Figure 2. 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.
  • Figure 1. 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.
  • 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.

Protocols

References

This product has been referenced in:

  • Batran RA  et al. The antianginal ranolazine mitigates obesity-induced nonalcoholic fatty liver disease and increases hepatic pyruvate dehydrogenase activity. JCI Insight 4:N/A (2019). Read more (PubMed: 30626749) »
  • Tong WH  et al. TLR-activated repression of Fe-S cluster biogenesis drives a metabolic shift and alters histone and tubulin acetylation. Blood Adv 2:1146-1156 (2018). Read more (PubMed: 29784770) »
See all 21 Publications for this product

Customer reviews and Q&As

1-10 of 16 Abreviews or Q&A

Answer

I reviewed details of the instrument on this page with the laboratories: http://info.bio-rad.com/ww-chemidoc-lp.html

It may work to read this assay, but to be sure the best would be to ask BioRad technical support to see if their instrument can read effectively lateral flow immunoassays.

I am sorry we don't have data to answer your question more definitely on this occasion but I hope this is helpful.

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Answer

There are a few different means of interpreting the results with our dipstick assays.

1) You can do relative quantification in which you do not need a dipstick reader. You would simply be visualizing the relative intensity and comparing between samples.

2) If you do not have a dipstick reader you can use an standard flatbed scanner to collect the images and then quantify using any densitometry software. There are many free densitometry software programs out there; we like Image J.

I hope this helps!

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Answer

I can confirm that either scrapping or enzymatic digestion with trypsin are acceptable methods for harvesting the cultured cells.

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Answer

We haven't tested the kit ab109882 with yeast samples so I am sorry we are unable to confirm the species cross reactivity.

None of the anti PDH antibody we have is tested with yeast sample so it is difficult for us to comment on the cross reactivity of these as well. In case you are interested in buying and trying few antibodies (e.g. ab110333, ab110334, ab110330 etc) then I would encourage you to participate in our 100% Abtrial discount programme - a free product can be claimed in exchange of review for antibodies. Please contact me for code if you accept our offer.

More details about the offer can be found at the following link;

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount

I would suggest doing a sequence alignment between human and S. cerevisiae protein to see if these is any compatibility. Please send me the aligned sequences I will then check the immunogen sequence of our antibodies for prediction of cross reactivity.

Please note we do not provide free small samples however we help our customers by providing Abtrial codes.

Read More

Answer

DCA or any other inhibitors of PDH kinases are not in any of the kit’s components. Also, no phosphatase inhibitors are present.

Read More

Answer

Thank you for your response.

You can use regular scanner to scan and analyze your result with image J software. I would suggest you to follow your plan, i.e. purchase a kit and check whether it is sensitive enough for your sample types.

In the future, if you require high quality results, you may need to consider to purchase a dipstick reader. Since a dipstick reader is a PC-based system for measuring and analyzing Abcam's dipstick products. Compare to regular scanner, the result generated by a dipstick reader is more sensitive and accurate with low CVs.

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for your response.

There is an instrument called "Dipstick Reader" which has been developed to read the signal. The Protocol Booklet and Measurement Program Instruction Manual can be downloaded through the website:

https://www.abcam.com/Dipstick-Reader-ab117066.html

All the available specific publications are also listed on the product datasheets (bottom section).

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for contacting us.

Please find attached the sample preparation guide we have.

10 million cells/ ml give 5 mg of protein/ ml. You can easily scale down the volume and number of cells.

You would only need 25 ul of samples per well at the suggested protein concentration.

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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Question
Answer

Thank you for your enquiry.

We have not tested Arabidopsis with this kit. The immunogen used to generate the capture antibody in this kit was Purified PDH protein from Pig Heart. Therefore, we are unable to predict homology with your species. If you wish to test the product, please refer to our Abtrial program.

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Answer

Thank you for contacting us.

With regards to ab109720, there is a publication on the utility of Complex I enzyme activity dipsticks in patients with specific mutations in the different subunits of complex I. The antibody stripped to the nitrocellulose is capable of capturing the multisubunit complex in its native form when the sample is extracted with the detergent included in the kit. The attached paper shows in detail how the antibodies were generated, how they were validated by mass spectrometry (see reference 14 for complex I) and how the dipstick activity kit performs on fibroblasts from patients with mutations in specific subunits of complex I.

With regards to ab109882 , the capture antibody used in the kit targets E2 – clone ID 15D3G9C11 (ab110332). IP characterization of this antibody was also published. The focused proteomics paper, also attached, shows how all subunits of PDH co-IP when using this antibody (in the paper is referred to as (15D3).

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 16 Abreviews or Q&A

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