Sausen 2019 Methods Mol Biol

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Sausen CW, Rogers CM, Bochman ML (2019) Thin-Layer Chromatography and Real-Time Coupled Assays to Measure ATP Hydrolysis. Methods Mol Biol 1999:245-253.

» PMID:31127581

Sausen Christopher W, Rogers Cody M, Bochman Matthew L (2019) Methods Mol Biol

Abstract: Many chemical reactions in the cell are thermodynamically unfavorable. To overcome this barrier, the energy released from the hydrolysis of adenosine triphosphate (ATP) is coupled to these reactions via ATP hydrolyzing enzymes known as ATPases. These enzymes are ubiquitous in nature and frequently act as molecular motors in processes ranging from DNA replication to protein degradation. Assays that characterize ATPase activity in vitro are important tools to gain insight into their functions in vivo. Here, we describe a direct and flexible thin-layer chromatography method for detecting ATPase activity using radiolabeled ATP. Additionally, we describe a high-throughput coupled reaction assay pairing ATP hydrolysis with nicotinamide adenine dinucleotide (NADH) oxidation to monitor ATP hydrolysis in real time.

Bioblast editor: Cardoso Luiza HD

Cited by

  • Cardoso et al (2021) Magnesium Green for fluorometric measurement of ATP production does not interfere with mitochondrial respiration. MitoFit Preprints 2021.1. doi:10.26124/mitofit:2021-0001


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