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Michaelis-Menten Kinetics Measurements of Aldo-Keto Reductases for Various Substrates in Murine Tissue.

STAR protocols (2020-12-31)
Jakob Morgenstern, Elisabeth Kliemank, Marta Campos Campos, Peter Nawroth, Thomas Fleming
RÉSUMÉ

Aldo-keto reductases (AKRs) are responsible for the detoxification of harmful aldehydes. Due to the large number of isotypes, the physiological relevance of AKRs cannot be obtained using mRNA or protein quantification, but only through the use of enzymatic assays to demonstrate functionality. Here, we present a fast and simple protocol to determine the important Michaelis-Menten kinetics of AKRs, which includes various aldehyde substrates of interest such as 4-hydroxynonenal, methylglyoxal, and malondialdehyde. For complete details on the use and execution of this protocol, please refer to Morgenstern et al. (2017) and Schumacher et al. (2018).

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Phosphate de sodium monobasic monohydrate, ACS reagent, ≥98%
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HEPES, ≥99.5% (titration)
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Cocktail d'inhibiteurs de protéases, for use with mammalian cell and tissue extracts, DMSO solution
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2-mercaptoéthanol, ≥99.0%
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Chlorure de magnésium solution, for molecular biology, 1.00 M±0.01 M
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Methylglyoxal solution, ~40% in H2O
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L-Glutathion réduit, ≥98.0%
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4-Hydroxynonenal, 4-Hydroxynonenal, CAS 75899-68-2, is a major aldehyde product formed by peroxidation of ω-6-unsaturated fatty acids that is regarded as a specific marker of lipid peroxidation.
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Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
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Phosphate de sodium dibasic, BioXtra, ≥99.0%
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Chlorure de potassium, BioXtra, ≥99.0%
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2-AAPA hydrate, ≥95% (HPLC)