ab109882 is used to quantify the activity of the PDH enzyme complex from human, bovine, mouse, rat and pig samples. This is accomplished by immunocapturing PDH with an anti-PDH antibody immobilized on a defined section of a dipstick. The enzyme complex is immunocaptured in its native form and activity is measured by the reaction scheme. PDH activity is visualized by coupling PDH-dependent production of NADH to the reduction of NBT in the presence of excess diaphorase, forming an insoluble intensely colored precipitate at the capture line. The signal intensity is measured by a dipstick reader or analyzed by other imaging systems such as a flatbed scanner.
Purified mitochondria can be used in this assay. However, homogenized tissue and whole cells can also be used without the need for mitochondrial isolation.
Pyruvate dehydrogenase activity is regulated by PDH kinase and PDH phosphatase. Cells grown in glucose media derive most of their energy from glycolysis; therefore, most of their PDH complex may be in an inactive phosphorylated form at the time of isolation. Growing cells in alternate carbon sources such as glutamine/galactose may up-regulate PDH activity. This kit does not include PDH kinases, PDH phosphatases or their respective inhibitors. These may be incorporated into the assay at the user’s discretion.
Store dipsticks at room temperature out of direct sunlight in their provided desiccant container. Avoid high humidity conditions.
Store Sample buffer at 4°C.
Store 20X Reagent Mix, Detergent, and Diaphorase at -80°C, but these may be aliquoted upon receipt to prevent freeze/thaw cycles.
Store Blocking solution at -20°C.
Store NBT in the dark at -80°C.
|Components||90 tests||30 tests|
|20X Reagent Mix||3 x 600µl||1 x 600µl|
|96-well microplate||6 units||2 units|
|Blocking solution||3 x 2ml||1 x 2ml|
|Detergent||3 x 1ml||1 x 1ml|
|Diaphorase||3 x 400µl||1 x 400µl|
|Dipsticks||1 x 90 units||1 x 30 tests|
|NBT||3 x 40µl||1 x 40µl|
|Sample buffer||3 x 30ml||1 x 30ml|
Our Abpromise guarantee covers the use of ab109882 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent dilution.|