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Complex I Enzyme Activity Assay Kit (ab109721) is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts.
Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the microplate wells which have been precoated with a specific capture antibody. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. 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.
Complex I activity is controlled by enzyme amount and by post-translational phosphorylation at key specific regulatory residues. Cellular metabolism governs these two factors. Ultimately, the cell type and growth conditions will affect Complex I activity measurements.
Note: This activity assay measures the diaphorase-type activity of Complex I. This activity is not dependant on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay. However, both the activity assay and the quantity assay described here are affected by enzyme assembly deficiencies.
ab109721 is shipped at 4°C. 20X NADH and 100X Dye are shipped lyophilized. Rehydrate 20X NADH by adding 1.1 mL H2O. Rehydrate 100X Dye by adding 0.25 mL H2O, then vortex each thoroughly until dissolved. After hydration unused amounts of these two materials should be stored at -80°C. When planning multiple experiments, aliquot these reagents to prevent freeze thaw cycles. Store all other components at 4°C
|100X Dye (lyophilized)||1 unit|
|10X Blocking Solution||1 x 10ml|
|10X Detergent||1 x 1ml|
|20X Wash Buffer||1 x 25ml|
|20X NADH (lyophilized)||1 unit|
|96-well microplate (12 strips)||1 unit|
Our Abpromise guarantee covers the use of ab109721 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.|