Application note: Promega’s ADP-Glo™ assay
This application note discusses how the ADP-Glo™ Kinase assay is a universal, homogeneous, high-throughput screening method that measures kinase activity by quantifying the amount of ADP produced during the kinase reaction.
Kinases are a large and diverse group of enzymes that are involved in many cellular metabolic and regulatory processes. During the kinase reaction, substrates are phosphorylated while ATP is converted into ADP. Due to the kinases’ pivotal role in intracellular signaling they are prominent drug targets.
The ADP-Glo™ Kinase assay is a universal, homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during the kinase reaction. It first eliminates remaining ATP and secondly converts ADP into ATP. The ATP is then quantified in an ATP-dependent light-emitting luciferase reaction.
To mimic an enzymatic reaction, an ADP/ATP standard curve was prepared from the nucleotide stock solutions that were supplied with the kit. Measuring the assay on a BMG LABTECH microplate reader revealed a signal stabilization around 20 min and that the instrument gives a linear signal from 1 mM down to >0.1 µM ADP.
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