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

概述

  • 产品名称Pyruvate dehydrogenase (PDH) Combo (Activity + Profiling) Microplate Assay试剂盒
    参阅全部 Pyruvate dehydrogenase 试剂盒
  • 检测方法Colorimetric
  • 样品类型
    Cell culture extracts, Tissue
  • 检测类型Enzyme activity
  • 实验步骤Multiple steps standard assay
  • 种属反应性
    与反应: Mouse, Rat, Cow, Human
  • 产品概述

    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.


     


     


     


     

  • 说明

    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.

  • 经测试应用适用于: Sandwich ELISAmore details
  • 平台Microplate

性能

  • 别名
    • PDC
    • PDH
    • PDHC

应用

Our Abpromise guarantee covers the use of ab110671 in the following tested applications.

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

应用 Ab评论 说明
Sandwich ELISA Use at an assay dependent dilution.

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

  • 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.
  • 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
  • Figure 1. PDH activity assay ab110671 reaction scheme.
  • 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 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.

实验方案

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

This product has been referenced in:
  • 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 1 Publication for this product

Product Wall



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: 250µ...

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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...

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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 recom...

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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...

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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...

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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. Read More

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 o...

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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 composit...

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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.

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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