Mitochondrial complex 1 activity assay kit

Manufactured by Merck Group

Sourced in Germany, United States

The Mitochondrial Complex I Activity Assay kit is a laboratory instrument designed to measure the activity of Complex I, also known as NADH-ubiquinone oxidoreductase, in biological samples. The kit provides the necessary reagents and protocols to quantify the specific enzymatic activity of this essential component of the mitochondrial electron transport chain.

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Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions) Microplate reader, by Bio-Rad (1 mentions) Hochest 33342, by Merck Group (1 mentions) Mitotracker green, by Thermo Fisher Scientific (1 mentions) Mammalian tissue lysis extraction reagent, by Merck Group (1 mentions)

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Activity Assay Kits

4 protocols using mitochondrial complex 1 activity assay kit

Assaying Mitochondrial Complex I Activity

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Mitochondrial complex I activity was measured using the Mitochondrial Complex I Activity Assay kit (Merck-Millipore, AAMT001-1KIT) according to the manufacturer's protocol. Cells and tissue were frozen and thawed 3 times as previously described⁴⁴ (link) and protein concentration was measured with the Bicinchoninic Acid Protein Assay kit (Pierce Biotechnology, 23225). Cell and tissue lysates were incubated with complex I capture antibody for 3 h at room temperature, and complex I activity was determined by measuring the oxidation of nicotinamide adenine dinucleotide (NAD)H to NAD⁺, which was observed as a reduction in the dye and a corresponding increase in absorbance at 450 nm after 30 min. Each sample was tested in duplicate and activity is expressed as the change in absorbance per minute per amount of sample loaded in the well.

Liu J., Liu W., Lu Y., Tian H., Duan C., Lu L., Gao G., Wu X., Wang X, & Yang H. (2018). Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models. Autophagy, 14(5), 845-861.

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Mitochondrial Complex I Activity Assay

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The Mitochondrial Complex I Activity Assay Kit determined the mitochondrial complex I activity of the IMCD3 cells (Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol. Mitochondrial complex I activity was detected at 450 nm absorbance using a microplate reader (Bio-Rad, USA).

Li Z., Zhou J., Li Y., Yang F., Lian X, & Liu W. (2020). Overexpression of DJ-1 alleviates autosomal dominant polycystic kidney disease by regulating cell proliferation, apoptosis, and mitochondrial metabolism in vitro and in vivo. Annals of Translational Medicine, 8(18), 1175.

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Mitochondrial Mass and Membrane Potential Assay

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For imaging mitochondrial mass or morphology independent of membrane potential [21 (link)], live cells were stained by MitoTracker Green (100 nmol·L^− 1, Invitrogen) at 37 °C for 30 min or transfected with MitoRFP reporter (Addgene) for 48 h, with nuclei staining by Hochest 33,342 (Sigma). Mitochondrial structure was observed by transmission electron microscopy [22 (link)]. Cells were treated with JC-1 (10 μg/ml, Sigma) at 37 °C for 20 min, while red fluorescence intensity reflecting Δψm was assessed with a fluorescence microscope. Mitochondrial complex I activity was determined by Mitochondrial Complex I Activity Assay Kit (Sigma).

Yang F., Hu A., Guo Y., Wang J., Li D., Wang X., Jin S., Yuan B., Cai S., Zhou Y., Li Q., Chen G., Gao H., Zheng L, & Tong Q. (2021). p113 isoform encoded by CUX1 circular RNA drives tumor progression via facilitating ZRF1/BRD4 transactivation. Molecular Cancer, 20, 123.

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Mitochondrial Complex I Activity Assay

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Mitochondrial complex I activity in SH-SY5Y cells exposed to pesticides was determined via Mitochondrial Complex I Activity Assay Kit (Sigma-Aldrich, Billerica, MA, USA, AAMT001). In brief, cells were grown in 10 cm dishes and exposed to pesticides, and then collected and lysed with Mammalian Tissue Lysis/Extraction Reagent (Sigma-Aldrich, Billerica, MA, USA, C3228). Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA, 23225). Mitochondrial complex I activity, determined as described in the manufacturer’s protocol, was defined as a change in absorbance at 450 nm per minute for each amount of sample. Each experiment was repeated three times.

Chen T., Tan J., Wan Z., Zou Y., Kessete Afewerky H., Zhang Z, & Zhang T. (2017). Effects of Commonly Used Pesticides in China on the Mitochondria and Ubiquitin-Proteasome System in Parkinson’s Disease. International Journal of Molecular Sciences, 18(12), 2507.

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