This rapid activity and quantity microplate kit is used to determine the activity and 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 quantity.
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 MitoSciences suggests using the MSP30 Dipstick Assay Kit for PDH Activity, which has a working range of 100-1000 µg depending on the cell type.
The quantity microplate is used to determine the 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.