Assay Principle

ADP-Glo Assay: In this assay, kinases interact with their substrates in the presence of ATP, which donates a phosphate group and produces ADP as a byproduct. After a set reaction time, an ADP-Glo reagent is added to stop the reaction and deplete remaining ATP, ensuring that only newly generated ADP is measured. A detection reagent then converts ADP back to ATP, which reacts with luciferase to produce a chemiluminescent signal proportional to kinase activity. 

Fluorescence Intensity (FI) Assay for NADP(H): This assay uses NADH/NADPH as a hydrogen carrier in the presence of a dehydrogenase enzyme, where it cycles between reduced (NADH/NADPH) and oxidized (NAD/NADP) forms. Upon excitation at 340 nm, NADH/NADPH emits fluorescence at 460 nm, while oxidized NAD/NADP forms do not emit. Fluorescence intensity directly reflects the concentration of NADH/NADPH, indicating the enzymatic activity. 

Absorbance (Abs) Assay: In this method, the substrate undergoes hydrolysis by the enzyme, producing a detectable product with a specific absorption peak. The optical density (OD) value of this product is measured using a spectrophotometer, providing a quantitative measure of enzyme activity based on absorbance changes.