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Product name
Complex I Enzyme Activity Microplate Assay Kit
See all Complex I products (7) ...
Tests
1 x 96 well plate
Sample type
Cell culture extracts, Tissue
Assay type
Enzyme activity
Cross reactivity
Reacts 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.
Notes
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 applications
Functional Studiesmore details
Properties
Storage instructions
Please see Notes section
| Components | 96 tests |
|---|---|
| 100X Dye (lyophilized) | 1 unit |
| 20X NADH (lyophilized) | 1 unit |
| 96-well microplate (12 strips) | 1 unit |
| Block (10X) | 1 x 10ml |
| Buffer (20X) | 1 x 25ml |
| Detergent (10X) | 1 x 1ml |
Applications
Show applications key
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.
FuncS
Complex I Enzyme Activity Microplate Assay Kit images:
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
Abcam's 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.
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
(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.
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (ab109721)
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.
Protocols
References for Complex I Enzyme Activity Microplate Assay Kit (ab109721)
This product has been referenced in:
- Favaro E et al. MicroRNA-210 regulates mitochondrial free radical response to hypoxia and krebs cycle in cancer cells by targeting iron sulfur cluster protein ISCU. PLoS One 5:e10345 (2010).Read more (PubMed: 20436681) »
- McIntosh R et al. The critical role of intracellular zinc in adenosine A(2) receptor activation induced cardioprotection against reperfusion injury. J Mol Cell Cardiol 49:41-7 (2010).Read more (PubMed: 20144616) »
See all 6 publications for this product
Publishing research using ab109721? Please let us know so that we can cite the reference in this datasheet
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"