Description
Key facts
Detection method:Colorimetric,
Sample types:Cell culture extracts, Tissue,
Reacts with:Mouse, Rat, Cow, Human,
Assay type:Enzyme activity,
Assay time:3h 30m,
Assay Platform:Microplate reader
Product details:
Mitochondrial Complex I enzyme activity assay principle
- capture antibodies specific for Complex I are pre-coated in the wells of the microplate included in the kit
- samples are added to the microplate wells
- Complex I is immobilized in the wells, and other proteins are then washed away
- active Complex I will oxidize NADH to NAD+, with the simultaneous reduction of a dye, leading 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.
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, the assay is affected by enzyme assembly deficiencies.
Mitochondrial Complex I enzyme activity assay protocol summary:
-Detergent extract of prepared samples loaded onto plate
- Incubate for 3 hours at room temperature. Wash plate wells with Buffer 3X
- Add 200 µL of Assay Solution to each well
- Measure Optical Density (OD450 nm) in kinetic mode at room temperature for up to 30 minutes
How other researchers are using
Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) has been used in a variety of sample type including:
Rat Whisker pad skin tissues
Human neuroblastoma cell line SK-N-SH
Human neuroglioma H4 cells
References:
1-Soma C et al. 2023
2-Fan W et al. 2023
3-Mc Gregor L et al. 2023
Related and recommended products
See other alternative kits to quantify other Complex enzymes activities:
- Complex II Enzyme Activity Microplate Assay Kit
ab109908
- Complex IV Rodent Enzyme Activity Microplate Assay Kit
ab109911
- Complex IV Human Enzyme Activity Microplate Assay Kit
ab109909
- Complex IV Human Specific Activity Microplate Assay Kit
ab109910
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions-+4°C, Appropriate long-term storage conditions-+4°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Complex I also known as NADH dehydrogenase or NADH:ubiquinone oxidoreductase is a large enzyme complex with a molecular mass of approximately 1000 kDa. It is expressed in the inner mitochondrial membrane of eukaryotic cells. As the first enzyme in the mitochondrial respiratory chain Complex I plays a critical mechanical role in cellular respiration. It transfers electrons from NADH to ubiquinone coupled with the translocation of protons across the inner mitochondrial membrane contributing to the generation of a proton gradient used to produce ATP.
Biological function summary
Complex I acts as an integral component of the mitochondrial respiratory chain which is a series of protein complexes involved in cellular energy production. As part of this complex system Complex I is essential for effective oxidative phosphorylation. Its activity is assessed using protein activity assays including immunocapture or complex activity assays and microplate assays. Complex I activity influences the overall efficiency of ATP production affecting energy-dependent cellular processes.
Pathways
Complex I functions within the electron transport chain one of the major pathways in cellular respiration. This pathway is vital for ATP synthesis providing the energy currency required by cells. Complex I works closely with other electron transport chain complexes such as Complex II (succinate dehydrogenase complex) and Complex III (cytochrome c reductase) to drive oxidation-reduction reactions and maintain cellular metabolism.
Defects in Complex I are linked with mitochondrial diseases and neurodegenerative disorders such as Leigh syndrome and Parkinson's disease. Mutations in Complex I subunits disrupt normal electron transport and ATP production leading to increased oxidative stress and neuronal cell damage. Other mitochondrial proteins such as cytochrome c oxidase are also implicated in these conditions emphasizing the interconnected nature of mitochondrial dysfunction in disease progression.
