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

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
摘要

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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Sigma-Aldrich
磷酸钠 一元 一水合物, ACS reagent, ≥98%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
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Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
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2-巯基乙醇, ≥99.0%
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氯化镁 溶液, for molecular biology, 1.00 M±0.01 M
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甲基乙二醛 溶液, ~40% in H2O
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L -还原型谷胱甘肽, ≥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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乙二胺四乙酸, anhydrous, BioUltra, ≥99% (titration)
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氯化钾, BioXtra, ≥99.0%
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磷酸钠 二元, BioXtra, ≥99.0%
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2-AAPA 水合物, ≥95% (HPLC)