ADP Accumulation Assays

Ideal Assays for Kinase Profiling and High-throughput Screening Applications

Insights into kinase function requires the determination of the binding affinity (Ki) and kinetics (Km) between the kinase and its substrate. These parameters, along with detecting the kinase activity and inhibition, are critical for kinase profiling and high throughput screening in drug discovery.

Eurofins DiscoverX® offers antibody-free, homogeneous ADP Hunter Plus and ADP Quest assays for evaluating kinase activity and inhibition directly through measurement of ADP accumulation. These ADP accumulation assays allow for identifying and characterizing kinase enzyme activity to transfer a phosphate from the phosphate-donating molecule ATP to its specific substrate. In contrast to standard assays that rely on antibody detection of a phosphoepitope or monitoring ATP depletion as the result of kinase activity, these assays are homogeneous gain-of-signal assays and do not require antibodies.

Results of these assays generate a positive readout in direct proportion to the ADP accumulation – outcomes from phosphotransferase activity. ADP Hunter Plus and ADP Quest assays are both kinetic and endpoint-mode-capable, providing the ability to determine the Km and Ki for insights into kinase function. In addition to evaluating ADP accumulation for kinases, these activity-based assays can also be applied to detect other nucleoside diphosphates such as UDP and GDP, and they have been broadly applied to other types of enzymes that utilize ATP, such as ATPases.

The results mean that with minimal steps, these Eurofins DiscoverX assays generate a fluorescence readout with a flexible assay read time and result in robust signal-to-background ratios. They offer excellent high DTT (dithiothreitol) and ATP tolerance and are compatible with unmodified peptides and whole protein substrates, making them ideal for enzyme profiling and high-throughput screening.