Complex I Enzyme Activity Assay Kit ab109721 is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity in human, rat, mouse and bovine cell and tissue extracts.
- Uses antibody capture method for superior specificity
- Cited in > 250 publications
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- Phytotherapy research : PTR 38:2496-25172024Quercetin inhibits necroptosis in cardiomyocytes after ischemia-reperfusion via DNA-PKcs-SIRT5-orchestrated mitochondrial quality control.Applications:Unspecified applicationReactive species:Unspecified reactive speciesXing Chang,Qin Zhang,Yu Huang,Jinfeng Liu,Yanli Wang,Xuanke Guan,Qiaomin Wu,Zhiming Liu,Ruxiu LiuPubMed 38447978
- Scientific reports 13:227602023Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats.Applications:Unspecified applicationReactive species:Unspecified reactive speciesChihiro Soma,Suzuro Hitomi,Eri Oshima,Yoshinori Hayashi,Kumi Soma,Ikuko Shibuta,Yoshiyuki Tsuboi,Tetsuo Shirakawa,Takashi Kikuiri,Koichi Iwata,Masamichi ShinodaPubMed 38123836
- Experimental and therapeutic medicine 27:402023Icariside II protects dopaminergic neurons from 1‑methyl‑4‑phenylpyridinium‑induced neurotoxicity by downregulating HDAC2 to restore mitochondrial function.Applications:Unspecified applicationReactive species:Unspecified reactive speciesWenbo Fan,Jianwu ZhouPubMed 38125349
- Nature communications 14:82482023The assembly of the Mitochondrial Complex I Assembly complex uncovers a redox pathway coordination.Applications:Unspecified applicationReactive species:Unspecified reactive speciesLindsay McGregor,Samira Acajjaoui,Ambroise Desfosses,Melissa Saïdi,Maria Bacia-Verloop,Jennifer J Schwarz,Pauline Juyoux,Jill von Velsen,Matthew W Bowler,Andrew A McCarthy,Eaazhisai Kandiah,Irina Gutsche,Montserrat Soler-LopezPubMed 38086790
- Journal of medical virology 95:e292702023SARS-CoV-2 nucleocapsid protein enhances the level of mitochondrial reactive oxygen species.Applications:Unspecified applicationReactive species:Unspecified reactive speciesHaiyun Yu,Lu Yang,Zhennan Han,Xiaoyu Zhou,Zihan Zhang,Tianli Sun,Fang Zheng,Jingzhi Yang,Fei Guan,Jungang Xie,Chaohong LiuPubMed 38047459
- Nature communications 14:76742023A reversible state of hypometabolism in a human cellular model of sporadic Parkinson's disease.Applications:Unspecified applicationReactive species:Unspecified reactive speciesSebastian Schmidt,Constantin Stautner,Duc Tung Vu,Alexander Heinz,Martin Regensburger,Ozge Karayel,Dietrich Trümbach,Anna Artati,Sabine Kaltenhäuser,Mohamed Zakaria Nassef,Sina Hembach,Letyfee Steinert,Beate Winner,Winkler Jürgen,Martin Jastroch,Malte D Luecken,Fabian J Theis,Gil Gregor Westmeyer,Jerzy Adamski,Matthias Mann,Karsten Hiller,Florian Giesert,Daniela M Vogt Weisenhorn,Wolfgang WurstPubMed 37996418
- JCI insight 8:2023Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.Applications:Unspecified applicationReactive species:Unspecified reactive speciesTing Yu,Shuke Nie,Lihong Bu,Miao Liu,Juanfeng He,Xuan Niu,Hongyan Feng,Jifeng Guo,Beisha Tang,Zhaohui Zhang,Keqiang Ye,Haiqiang Jiang,Liam Chen,Zhentao ZhangPubMed 37937646
- Scientific reports 13:171762023NARFL deficiency caused mitochondrial dysfunction in lung cancer cells by HIF-1α-DNMT1 axis.Applications:Unspecified applicationReactive species:Unspecified reactive speciesHongzhou Liu,Xueqin Wu,Tianrong Yang,Chen Wang,Fei Huang,Ying Xu,Jie PengPubMed 37821486
- Molecular metabolism 77:1018022023Partial skeletal muscle-specific Drp1 knockout enhances insulin sensitivity in diet-induced obese mice, but not in lean mice.Applications:Unspecified applicationReactive species:Unspecified reactive speciesBenjamin A Kugler,Jared Lourie,Nicolas Berger,Nana Lin,Paul Nguyen,Edzana DosSantos,Abir Ali,Amira Sesay,H Grace Rosen,Baby Kalemba,Gregory M Hendricks,Joseph A Houmard,Hiromi Sesaki,Philimon Gona,Tongjian You,Zhen Yan,Kai ZouPubMed 37690520
- Nature communications 14:54962023The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.Applications:Unspecified applicationReactive species:Unspecified reactive speciesSimeon R Mihaylov,Lydia M Castelli,Ya-Hui Lin,Aytac Gül,Nikita Soni,Christopher Hastings,Helen R Flynn,Oana Păun,Mark J Dickman,Ambrosius P Snijders,Robert Goldstone,Oliver Bandmann,Tatyana A Shelkovnikova,Heather Mortiboys,Sila K Ultanir,Guillaume M HautberguePubMed 37679383
Key facts
- Detection method
- Colorimetric
- Sample types
- Cell culture extracts, Tissue
- Assay type
- Enzyme activity
- Reactive species
- Mouse, Rat, Cow, Human
- Assay time
- 3h 30m
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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 in human, rat, mouse and bovine cell and tissue extracts.
- Uses antibody capture method for superior specificity
- Cited in > 250 publications
Key facts
- Detection method
- Colorimetric
- Sample types
- Cell culture extracts, Tissue
- Assay type
- Enzyme activity
- Reactive species
- Mouse, Rat, Cow, Human
- Assay time
- 3h 30m
- Assay Platform
- Microplate reader
Storage
- Shipped at conditions
- Blue Ice
- Appropriate short-term storage conditions
- +4°C
- Appropriate long-term storage conditions
- +4°C
- Storage information
- +4°C
Notes
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 1
Human neuroblastoma cell line SK-N-SH 2
Human neuroglioma H4 cells 3
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 Complex II Enzyme Activity Microplate Assay Kit ab109908
- Complex IV Rodent Enzyme Activity Microplate Assay Kit Complex IV Rodent Enzyme Activity Microplate Assay Kit ab109911
- Complex IV Human Enzyme Activity Microplate Assay Kit Complex IV Human Enzyme Activity Microplate Assay Kit ab109909
- Complex IV Human Specific Activity Microplate Assay Kit Complex IV Human Specific Activity Microplate Assay Kit ab109910
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
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.
- Associated diseases and disorders
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.
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6 product images
Image courtesy of Yao X et al.PLoS One. 2015; 10(10): e0139416. doi: 10.1371/journal.pone.0139416.Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)
Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.
Mitchondiral fractions from the heart tissue of Rats infected by S. pneumoniae, or given PBS sham control, were subjected to measurements of complex I-V activities. Complex I was measured with ab109721 (top left), Complex II + III were measured using MitoTox™ Complex II + III OXPHOS Activity Assay Kit ab109905 (top right), Complex IV was measured using Complex IV Rodent Enzyme Activity Microplate Assay Kit ab109911 (bottom left) and Complex V was measured using ATP synthase Enzyme Activity Microplate Assay Kit ab109714 (bottom right).
Freshly isolated mitochondrial pellets were resuspened in PBS supplemented with 10% detergent provided in the kits. Protein concentrations of these mitochondrial lysates were estimated and 25 μg (for complex I, IV and V) or 100 μg (for complex II+III) mitochondrial protein was used per reaction. Enzyme activities were measured spectrophotometricly in triplicate and expressed as changes of absorbance per minute per mg protein.
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)
ab109721 measures Complex I activity in human cultured cells within the recommended ranges given in the protocol. Example of Complex I activity measured in Hep2 cells is showed. (Note that these ranges depend on mitochondria preparation quality).
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (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. Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)
Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)
(A) ab109721 was used to measure Complex I activity in normal and Rho0 human fibroblast whole cell lysates at 1mg/mL. Rho0 cells are cells in which the mitochondrial DNA has been removed and therefore essential Complex I proteins are not expressed. As shown, the rho0 cells showed no/little complex I activity.
(B) In a similar analysis, 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 in samples loaded at 0.5 mg/mL whole cell lysates.
Functional Studies - Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)
Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)
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