Complex I Enzyme Activity Microplate Assay Kit (ab109721)
- Product nameComplex I Enzyme Activity Microplate Assay KitSee all Complex I kits ...
- Detection methodColorimetric
- Tests1 x 96 well plate
- Sample typeCell culture extracts, Tissue
- Assay typeEnzyme activity
- Assay durationMultiple steps standard assay
- Species reactivityReacts with: Mouse, Rat, Cow, Human
- Product overview
A 96-well microplate assay that is used to determine the activity of Complex I in a human, mouse or rat sample. The enzyme is immunocaptured within the wells of the microplate and activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at 450 nm.
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
- Tested applicationsFunctional Studies more details
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 96 tests 100X Dye (lyophilized) 1 unit 10X Blocking Buffer 1 x 10ml 10X Detergent 1 x 1ml 20X NADH (lyophilized) 1 unit 96-well microplate (12 strips) 1 unit Wash Buffer (20X) 1 x 25ml
- Research Areas
- NADH dehydrogenase
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.
Complex I Enzyme Activity Microplate Assay Kit images
ab109721 measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples within the recommended ranges given in the protocol. (Note that these ranges depend on mitochondria preparation quality.) Examples of Complex I activity measured in different rat and mouse mitochondrial samples are shown in the graphs above.
(A) ab10972 was used to determine the Complex I activity in normal fibroblasts and rho0 fibroblasts (cells in which the mtDNA is removed and hence essential Complex I proteins are not expressed). The rho0 cells showed no/little complex I activity. In a similar analysis, (B) rat cardiomyocytes were grown for 5 days in ± 40 µM chloramphenicol (CAM) to inhibit mitochondrial protein synthesis, Complex I assembly and hence activity was greatly reduced.
Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)
References for Complex I Enzyme Activity Microplate Assay Kit (ab109721)
This product has been referenced in:
- Kumarasamy S et al. Construction of two novel reciprocal conplastic rat strains and characterization of cardiac mitochondria. Am J Physiol Heart Circ Physiol 304:H22-32 (2013). Rat . Read more (PubMed: 23125210) »
- van den Bosch BJ et al. Defective NDUFA9 as a novel cause of neonatally fatal complex I disease. J Med Genet 49:10-5 (2012). Functional Studies . Read more (PubMed: 22114105) »