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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
RESUMO

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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Fosfato de sódio, ACS reagent, ≥98%
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HEPES, ≥99.5% (titration)
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Coquetel Inibidor de Protease, for use with mammalian cell and tissue extracts, DMSO solution
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2-Mercaptoetanol, ≥99.0%
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Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
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Methylglyoxal solution, ~40% in H2O
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L-Glutationa reduzida, ≥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, BioUltra, anhydrous, ≥99% (titration)
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Cloreto de potássio, BioXtra, ≥99.0%
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Fosfato de sódio, BioXtra, ≥99.0%
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2-AAPA hydrate, ≥95% (HPLC)