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Merck

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
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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 etย al. (2017) and Schumacher etย al. (2018).

MATERIALS
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2-Mercaptoethanol, ≥99.0%
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Magnesium chloride solution, for molecular biology, 1.00 Mยฑ0.01 M
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Potassium chloride, BioXtra, ≥99.0%
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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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Sodium phosphate monobasic monohydrate, ACS reagent, ≥98%
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Ethylenediaminetetraacetic acid, BioUltra, anhydrous, ≥99% (titration)
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L-Glutathione reduced, โ‰ฅ98.0%
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Methylglyoxal solution, ~40% in H2O
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Sodium phosphate dibasic, BioXtra, ≥99.0%