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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
ABSTRACT

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).

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2-Mercaptoethanol, ≥99.0%
Sigma-Aldrich
Protease Inhibitor Cocktail, for use with mammalian cell and tissue extracts, DMSO solution
Sigma-Aldrich
Potassium chloride, BioXtra, ≥99.0%
Sigma-Aldrich
HEPES, ≥99.5% (titration)
Sigma-Aldrich
Magnesium chloride solution, for molecular biology, 1.00 M±0.01 M
Sigma-Aldrich
Sodium phosphate monobasic monohydrate, ACS reagent, ≥98%
Sigma-Aldrich
L-Glutathione reduced, ≥98.0%
Sigma-Aldrich
Ethylenediaminetetraacetic acid, anhydrous, BioUltra, ≥99% (titration)
Sigma-Aldrich
Methylglyoxal solution, ~40% in H2O
Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
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.
Sigma-Aldrich
2-AAPA hydrate, ≥95% (HPLC)