ADP Hunter Plus Kit From DiscoveRx

Kinase assays have been one of the most important tools in the study of biology due to the enormous number of kinases which are directly linked with a variety of disease states. The discovery of kinase inhibitors for specific diseases holds great promise for the pharmaceutical industry and thus development of kinase assays has garnered a lot of attention.

In a kinase reaction, a phosphate molecule is transferred from high-energy donor molecules like ATP to a substrate and an ADP molecule is generated as a result of the reaction. Kinases are involved in signal transduction machinery as well as a lot of complex cell-biological processes. Different research groups have tried to analyze the outcome of kinase reactions by employing different methodologies including: depletion of ATP concentration, radioactive incorporation, downstream signaling output as a direct consequence of the kinase reaction, just to name a few.

The ADP Hunter Plus Kit from DiscoveRx Corporation is based on a much simplified methodology and uses fluorometric detection of ADP which is generated at the end of a kinase reaction. Other than the ADP Hunter assay, DiscoveRx offers a broad portfolio of different kinase solutions, including tools for functional activity, screening, profiling and pathway analysis. In the case of the ADP Hunter assay, the principle of detection is straightforward and simple. This assay directly detects the ADP molecule generated as a result of a phosphorylation reaction employing a non-radioactive, non-antibody based method. With the proprietary reagents included in the kit, the ADP molecule that is generated as a result of a kinase reaction is detected in the form of a “gain of function” approach. A coupled enzyme reaction system is used to generate hydrogen peroxide from ADP. Hydrogen peroxide when combined with ADHP (fluorescent dye precursor) in the presence of peroxidase generates the fluorescently active resorufin dye which is detected with a fluorescent plate reader with excitation and emission maxima being at 530 nm and 590 nm, respectively. (This reaction is explained schematically in the following figure; figure obtained from DiscoveRx with kind permission).

The assay design is compatible with 384-well plates and can definitely be scaled to other microtiter plates for HTS applications. This assay is easy to use and is specifically designed for screening of kinase inhibitors.

I have used this kit from DiscoveRx with outstanding success. I used purified recombinant protein for my assays. An ADP standard curve was generated for calculating the unknown concentrations. My assays were run on 384-well plates and the data obtained using a BioTek Synergy4 plate reader was very accurate. I could study ATP uptake (as a function of time) on my protein using this reaction kit with a standard substrate. Autophosphorylation activity of my protein was also analyzed using the ADP Hunter kit and it has generated good data.

The kinase reaction needs to reach equilibrium before addition of the reagents, unless a time-course study is being employed. Sequential addition of the Reagents A and B is a very important step in the process. Addition of the stop solution at the end of incubation with reagents A&B ensures signal stability and consistency. The manufacturer’s instructions, which come with the kit, are user-friendly. The reaction volume and reagents’ volume can be scaled per the requirements of the researcher.

The assay system works efficiently and I have not faced any major problems as such. The only downside is that the reagents are proprietary and cannot be made in the lab. The kit is a bit on the expensive part of the economic spectrum.