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Activity Based ADP Accumulation Assay Platform

Activity based ADP accumulation assay is a  generic, antibody-free homogeneous assay platform 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 measure the generation of ADP as the result of phosphotransferase activity, generating a positive readout in direct proportion to ADP accumulated.

Technology Principle

The detection of ADP is based on an enzyme-coupled reaction converting ADP to a fluorescent signal. The assays are simple and homogeneous and are ideal for both high throughput screening and kinase profiling applications. The assays have an excellent ATP tolerance and robust signal to background ratios, and compatible with unmodified peptides and whole protein substrates. The assay 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.


For every substrate molecule phosphorylated, an ADP molecule is generated.  The ADP assays measure ADP generated in such reactions using a coupled-enzyme reaction to convert ADP into hydrogen peroxide, which when combined with a fluorescent dye precursor and peroxidase, generates fluorescent resorufin.

Assay Performance Metrics

PKA and Jnk2

 

PKA

  
Kinase activity curves for PKA and Jnk2 in the presence of 25 µM ATP.  The assay produces robust windows and is applicable for kinases with a broad range of activity.  The assays high tolerance for ATP permit the determination of compound activity at or above the ATP Km [click graph to enlarge].   Staurosporine and H-7 inhibition of PKA kinase activity in the presence of 25 µM ATP.  Kinase reactions were performed at 30°C for 60 minutes.  Data shown was normalized as percentage signal change [click graph to enlarge].

Features & Benefits

  • Capability to run kinetic and endpoint assays
  • Can be used with a variety of phosphotransferases, such as kinases, ATPases, UTPases, GTPases
  • Compatible with peptide substrates and whole proteins
  • Homogeneous, non-radioactive format