Pyruvate dehydrogenase (PDH) Combo (Activity + Profiling) Microplate Assay Kit (ab110671)

Overview

  • Product name

    Pyruvate dehydrogenase (PDH) Combo (Activity + Profiling) Microplate Assay Kit
    See all Pyruvate dehydrogenase kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture extracts, Tissue
  • Assay type

    Enzyme activity
  • Species reactivity

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

    This rapid activity and quantity microplate kit is used to determine the activity and relative quantity of PDH in a human, bovine, mouse or rat sample. This microplate assay kit includes two microplates, one to determine the PDH activity of your sample and one to determine the PDH relative quantity levels.


    The activity microplate 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 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. Therefore for studies with cultured cells Abcam suggests using the Dipstick Assay Kit for PDH Activity (ab109882/MSP30) which has a working range of 100-1000 µg depending on the cell type.


    The profiling microplate is used to determine the relatvie amount of PDH in a human, bovine, mouse, or rat sample with speed and simplicity. The PDH enzyme is immunocaptured within the wells of the microplate, and the amount is determined by adding a PDH specific antibody conjugated with alkaline phophatase. This phosphatase changes the substrate pNPP from colorless to yellow (OD 405 nm) in a time dependent manner proportional to the amount of protein captured in the wells. Included for performance in the quantity assay are buffer, detergent, antibodies, substrates, and a 96-well microplate with monoclonal antibody pre-bound to the wells of the plate, allowing for a stream-lined assay.


     


     


     


     

  • Notes

    Store 20X Reagent Mix and 100X Coupler at -80°C. Store 5X Stabilizer and 100X Reagent Dye at -20°C. Store all other components at 4°C.

  • Platform

    Microplate reader

Properties

Images

  • Figure 4. An example of the quantity of PDH capture from a HepG2 cultured cell lysate. The sample was diluted to show that over this range of concentrations that can be used. Each sample was measured in 6 replicates. Bars show standard deviations.

    Figure 4. An example of the quantity of PDH capture from a HepG2 cultured cell lysate. The sample was diluted to show that over this range of concentrations that can be used. Each sample was measured in 6 replicates. Bars show standard deviations.
  • Figure 3. 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.
  • Figure 1. PDH activity assay ab110671 reaction scheme.
  • Abcams' protein quantity microplate assays use the well-established sandwich ELISA format, whereby capture and detector antibodies are used to immobilize and then quantify a target protein or enzyme. All of our microplate assays utilize our highly-validated immunocapture antibodies, which are able to capture large, multi-subunit enzyme complexes in their fully intact state. Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, a second monoclonal antibody, against a different epitope on the target, is added to the well. This detector antibody is either directly labeled with biotin, or a biotin-labeled goat anti-mouse secondary is added. Substrate plus HRP or AP conjugated to streptavidin provide a colorimetric signal that is readable by any plate readers capable of standard ELISA absorbance measurements
  • Abcams' enzyme activity assays apply a novel approach, whereby target enzymes are first immunocaptured from tissue or cell samples before subsequent functional analysis. 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. Capture antibodies are pre-coated in the wells of premium Nunc MaxiSorp™ modular microplates, which can be broken into 8-well strips. After the target has been immobilized in the well, substrate is added, and enzyme activity is analyzed by measuring the change in absorbance of either the substrate or the product of the reaction (depending upon which enzyme is being analyzed). By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.

Protocols

References

This product has been referenced in:

  • Zhang Y  et al. miR-378 Activates the Pyruvate-PEP Futile Cycle and Enhances Lipolysis to Ameliorate Obesity in Mice. EBioMedicine 5:93-104 (2016). Read more (PubMed: 27077116) »
  • Halim ND  et al. Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons. Glia 58:1168-76 (2010). Read more (PubMed: 20544852) »
See all 2 Publications for this product

Customer reviews and Q&As

1-10 of 20 Abreviews or Q&A

Question
Answer



I've heard back from the lab, and the full micrplate specifications are as follows:

Thermo Scientific; Nunc; Immuno Module Plate; PS; Framed; 96-wells/frame; C8; MaxiSorp surface; Total vol: 350µL/well; Working vol: 250µL/well; Surface area: 2.5cm2/well; Dim.: 128 x 86mm.

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Answer

To determine the amount of NADH oxidized by PDH, we need to convert the color change of the dye to NADH oxidized. These react in a 1:1 ratio.

The molar extinction for the dye is 25.9/ mM / well

So divide the increase in absorbance in each well at 450 nm by 25.9 to determine mM NADH oxidized in that period of time.

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Answer

Thank you for contacting Abcam.

If you want to measure endogenous PDH activity we recommend using both phosphatase inhibitors as well as ATP depletion system in the homogenization buffer, in addition to phosphatase inhibitor cocktail. We recommend supplementing PBS with 20 mM NaF and 100x diluted P8340. The P8340 is protease not phosphatase inhibitor cocktail.

For parallel measurement of activity and quantity, I would recommend to prepare one sample (in the presence of all 3 types of inhibitors) and then dilute the extract according the recommended range for these two assays. The phosphatase inhibitors and the ATP depletion system are not required for the quantity assay, unless phosphoserine antibodies are used as detectors.

If there is anything else I can help you with, please let me know.

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Answer

Thank you for your reply. I am sorry for the confusion.

The protocols are ultimately measuring similar things. What is different is the platform (two assays to hopefully come to the same conclusion). Protocol 1 is an immunocapture activity assay, whereas protocol #2 is a sandwich ELISA type of assay measuring specifically the post-translational modification. So the first protocol will give an answer such as “decrease PDH activity as a kinetic measurement of the enzyme substrate uptake” whereas the second one will give an answer of “increase PDH phosphorylation at a specific site”

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

Thank you for contacting us. While many of the steps of both protocols are similar there are differences in some of the reagents used and in a few of the steps as the two parts of the kit are collecting different data. While both parts immunocapture PDH, ab110174 (2nd part: quantity) will measure total PDH using a secondary antibody whereas ab109902 (1rst part: activity) will measure enzyme activity by measuring reductionNAD+ to NADH, coupledto the reduction of a reporter dye. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews

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Answer

Thank you for contacting Abcam.

You should be ok to use frozen tissue samples, one point to keep in mind though is that you always want to make sure you compare frozen to frozen samples and not fresh to frozen. Otherwise you should be good.

Also, I would not think of the Phosphatase inhibitor solution as 25x, I would just add the stated volumes and you not have any issues.

If there is anything else I can help you with, please let me know.

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Question
Answer

Thank you for contacting us.

The capture antibody on the plate is raised against porcine pyruvate dehydrogenase E2/E3bp.

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

Thank you for your reply. This kit is a combination of two of our other kits, our PDH Enzyme Activity Microplate Assay Kit (ab109902)and our PDH Protein Quantity Microplate Assay Kit (ab110174). The 20x Buffer in these kits are of identical composition with the only difference being size. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information. Use our products? Submit an Abreview. Earn rewards! https://www.abcam.com/abreviews

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Question
Answer

Thank you for calling Abcam yesterday. I also wanted to give you the email address of your representative, it is. Please let me know if there is anything else I can help you with.

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Question
Answer

Thank you for calling Abcam yesterday.
The 20X Buffer recipe is as follows:


Please let me know if there is anything else I can help you with.

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1-10 of 20 Abreviews or Q&A

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