Metabolic pathways are fundamental to cellular function, providing the energy and building blocks necessary for growth, maintenance, and responses to environmental changes. Dysregulation of metabolic enzymes and pathways is linked to a variety of diseases, including cancer, metabolic disorders, and neurodegenerative diseases. Targeting these pathways through drug discovery enables the development of therapies designed to correct metabolic imbalances or exploit metabolic vulnerabilities in diseased cells.
Kinases interact with their substrates in the presence of ATP, which donates a phosphate group and produces ADP as a byproduct. After a defined reaction time, ADP-Glo reagent stops the reaction and depletes remaining ATP so that newly generated ADP can be measured. A detection reagent then converts ADP back to ATP, which reacts with luciferase to produce a chemiluminescent signal proportional to kinase activity.
This Fluorescence Intensity (FI) assay uses NADH or NADPH as a hydrogen carrier in the presence of a dehydrogenase enzyme. NADH/NADPH emits fluorescence at 460 nm after excitation at 340 nm, whereas the oxidized NAD/NADP forms do not emit. Fluorescence intensity therefore reflects the concentration of NADH/NADPH and indicates enzymatic activity.
In the absorbance assay, enzymatic hydrolysis produces a detectable product with a specific absorption peak. The optical density value is measured using a spectrophotometer, providing a quantitative measure of enzyme activity based on changes in absorbance.
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