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

  Functional Studies - Pyruvate dehydrogenase (PDH) Combo (Activity + Quantity) Microplate Assay Kit (ab110671)

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.

  Sandwich ELISA - Pyruvate dehydrogenase (PDH) Combo (Activity + Quantity) Microplate Assay Kit (ab110671)

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

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

Figure 1. PDH activity assay ab110671 reaction scheme.

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

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.

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

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.