ADP Accumulation Assays

Ideal Assays for Kinase Profiling and High Throughput Screening Applications
 

The DiscoverX ADP accumulation assays are generic, antibody-free, homogeneous assays that detect kinase activity and inhibition by directly measuring ADP accumulation. DiscoverX’s ADP Hunter™ Plus and ADP Quest™ Assays are both kinetic and end-point mode capable, providing the ability to determine Km and Ki for insights into kinase function. The assays have minimal addition steps and generate robust fluorescence readout which allows for flexible assay read time. The ADP accumulation assays are compatible with proteins and peptide substrates, and are tolerant to high DTT (dithiothreitol) and ATP, making them ideal for kinase profiling and high throughput screening.


 

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

Products

View the available fluorescence ADP accumulation assays.
ADP Accumulation Assays

Ultrapure ATP for optimal assay performance.
ATP Gold

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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.