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.
- Multimode Readout – Kinetic and endpoint modes ideal for inhibitor screening and profiling
- User Friendly – Homogeneous, antibody-free assays with a fluorescence gain-of-signal readout allowing for flexible assay read time
- Universal Activity Assays – Versatile assays to detect enzyme activity and inhibition for any phosphotransferases, such as ATPases, UTPases, or GTPases.
- ADP Accumulation Assays – Homogeneous, antibody-free, and kinetic or end-point assays ideal for kinase profiling and high-throughput screening applications
- ATP Gold – Ultrapure ATP for optimal performance with ADP Hunter Plus and ADP Quest assays
- Recombinant Kinases – Largest portfolio of purified recombinant proteins for screening inhibitors
Activity-Based ADP Accumulation Assays
ADP Hunter Plus and ADP Quest assays are homogeneous antibody-free assays for measuring ADP accumulation that are ideal for kinase profiling and high throughput screening applications. The ADP Hunter Plus assay is optimized for high throughput screening, compatible with reducing agents such as DTT, and applicable for endpoint detection of ADP. In contrast, the ADP Quest Assay is an ideal assay for kinase and inhibitor characterization to provide a fast, accurate tool for determining Km, Ki, and mode-of-action, and applicable for kinetic or endpoint mode.
The assay uses enzymatic reactions that produce a signal that is directly proportional to the amount of ADP in the solution. These reactions utilize ADP to convert ADHP (10-Acetyl-3,7-dihydroxyphenoxazine), a fluorescent dye precursor, to fluorescent dye resorufin.
Gain Insight into Enzymology – Determine Kinase ATP Km
Determination of Kinase ATP Km. Michaelis-Menten plot for determining the ATP Km (Michaelis-Menton constant) for protein kinase A (PKA) which provides insight into enzymology and mode of inhibition. This assay has Km = 23 µM and R2 = 0.990.
Characterize Kinase Activity in Both Kinetic and Endpoint Modes
Kinetic and endpoint modes kinase activity characterization.
– (A.) PKA activity was measured at various concentrations with fixed substrate and ATP concentrations in kinetic mode. Assays in kinetic mode allows for real-time assay optimization. Note that at higher enzyme concentrations the substrate is depleted and a plateau is reached as expected.
– (B.) PKA and JNK2α2 kinases were incubated with kemptide and ATF-2, respectively and their activities were titrated in the presence of 25 µM ATP. The ADP accumulation assay shows a high tolerance for ATP and produces a robust assay window. This allows the assay to be applicable for kinases with a broad range of activity and for the determination of compound activity at or above the ATP Km.
Profile Kinase Inhibitors
Profiling protein kinase A (PKA) inhibitors. Staurosporine (SS) and H-7 inhibition of PKA (10 ng/mL) activity in the presence of 25 µM ATP. IC50 values for staurosporine and H-7 for PKAα are consistent with the literature and demonstrate the ability of the assay to identify weak inhibitors (normalized as percentage signal change in endpoint mode).
Rank Order Kinase Activity in the Presence of an Inhibitor
Rank ordering of kinase activity. Staurosprine was titrated for three kinases (PKA, JNK2α2, and CK1) with peptide substrates using the endpoint procedure. Peptide substrates included 100 μM kemptide for PKA, 250 μM CK peptide for CK1, and 100 μM ATF2 peptide for JNK2α2, and ATP concentrations was held constant at their respective Km concentrations (25 μM for PKA, 50 μM for CK1, and 100 μM for JNK2α2). The assay correctly represents the rank order of activity of the three kinases (PKA > CK1 > JNK2α2) and suggests that staurosporine is a potent inhibitor of PKA and a weak inhibitor for both CK1 and JNK2α2.
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