Activity-Based ADP Accumulation Assays
Easy-to-use, Kinetic and Endpoint Mode Capable
Activity based ADP accumulation assays are generic, antibody-free, and homogeneous – ideal for identifying and characterizing phosphotransferase activity. In contrast to standard assays which rely on antibody detection of a phospho-epitope or monitoring ATP depletion as the result of kinase activity, these assays are gain-of-signal assays that generate a positive readout in direct proportion to ADP as a result of phosphotransferase activity.
The assay uses an enzyme-coupled reaction that produces a red-shifted fluorescence signal that is directly proportional to the amount of ADP in the solution. The enzyme-coupled reaction utilizes ADP to generate hydrogen peroxide which in turn combines with a fluorescent dye precursor (ADHP) and peroxidase to generate fluorescent resorufin.
Kinase Assay Options
The ADP Quest assay is ideal for kinase and inhibitor characterization
- Fast and accurate tool for determining Km, Ki, and mode of action
- Application for kinetic or endpoint mode
The ADP Hunter assay is optimized for high throughput screening
- Compatible with reducing agents such as DTT
- Applicable for endpoint detection of ADP
View the available fluorescence ADP accumulation assays.
ADP Accumulation Assays
Ultrapure ATP for optimal assay performance.
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ADP Hunter™ Plus and ADP Quest™ Assays Highlights
ADP accumulation assays are simple and homogeneous, ideal for both high throughput screening and kinase profiling applications. They have an excellent ATP tolerance and robust signal-to-background ratios, and are compatible with unmodified peptides and whole protein substrates. These assays can also be applied to detect other NDPs such as UDP and GDP, and have been broadly applied to other types of enzymes that utilize ATP, such as ATPases.
Kinetic and end-point modes ideal for inhibitor screening and profiling
Robust fluorescence readout allows for flexible assay read time
Easy-to-use and universal for any phosphotransferases, such as ATPases, UTPases, GTPases
Gain Insight into Enzymology - Ideal Assays for Determining the Kinase ATP Km
Michealis-Menten plot for determining the ATP Km (Michealis-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 Mode and Endpoint Mode
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 activity 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 determination of compound activity at or above the ATP Km
Easily Profile Inhibitors
Staurosporine (SS) and H-7 inhibition of PKA kinase (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
Staurosprine was titrated for three kinases, PKA, JNK2α2, and CK1 with the following peptide substrates: 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) using the endpoint procedure. 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.