Product namePyruvate dehydrogenase (PDH) Combo (Activity + Profiling) Microplate Assay Kit
See all Pyruvate dehydrogenase kits
Sample typeCell culture extracts, Tissue
Assay typeEnzyme activity
Species reactivityReacts with: Mouse, Rat, Cow, Human
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.
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.
Storage instructionsPlease refer to protocols.
Components 1 x 96 tests 10X Blocking Solution 1 x 10ml 20X Detector Antibody 1 x 1ml 20X HRP Label 1 x 1ml 20X Reagent Mix 2 x 600µl 5X Stabilizer 2 x 13ml 96-well Microplate (12 strips) 2 units ab109902 20X Buffer 1 x 15ml ab110174 20X Buffer 1 x 20ml Coupler 1 x 250µl Detergent 3 x 1ml HRP/TMB Development Solution 1 x 20ml Reagent Dye 1 x 250µl
- Bovine Heart Mitochondria (ab110338)
- Rat liver tissue lysate - mitochondrial extract (ab110346)
- Rat heart tissue lysate - mitochondrial extract (ab110347)
- Rat brain tissue lysate - mitochondrial extract (ab110348)
- Mouse liver tissue lysate - mitochondrial extract (ab110349)
- Mouse heart tissue lysate - mitochondrial extract (ab110350)
- Mouse brain tissue lysate - mitochondrial extract (ab110351)
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.
ab110671 has been referenced in 3 publications.
- Bolfer L et al. Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts. Sci Rep 10:3930 (2020). PubMed: 32127618
- 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). 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). PubMed: 20544852