ADP-Glo™ Max Assay

The ADP-Glo™ Max Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure ATPase or kinase activity by quantifying the amount of ADP produced in a reaction. The assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) when higher ATP concentration is required (up to 5mM). The ADP-Glo™ Max Assay produces a strong signal that positively correlates with enzyme activity and can be adapted to a multitude of plate formats.

The assay is performed in two steps: first, after the completion of the ADP...

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The ADP-Glo™ Max Assay is a luminescent ADP detection assay that provides a universal, homogeneous, high-throughput screening method to measure ATPase or kinase activity by quantifying the amount of ADP produced in a reaction. The assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) when higher ATP concentration is required (up to 5mM). The ADP-Glo™ Max Assay produces a strong signal that positively correlates with enzyme activity and can be adapted to a multitude of plate formats.

The assay is performed in two steps: first, after the completion of the ADP-producing reaction, an equal volume of ADP-Glo™ Reagent is added to terminate the reaction and deplete the remaining ATP. Second, the ADP-Glo™ Max Detection Reagent is added to simultaneously convert ADP to ATP, and the latter is converted to light in a coupled reaction with luciferase/luciferin.

The ADP-Glo™ Max Assay has a high dynamic range and produces a strong signal at low ATP to ADP conversion, making it well suited for screening low-activity ATPases such as drug membrane transporters and heat shock proteins. The assay produces minimal false hits and Z´ values of greater than 0.7.

Several Kinase Enzyme Systems are available. Visit www.promega.com/kinase/ to see the collection.

Features - Benefits

• High Signal Strength at Low ATP Conversion: Users can measure enzyme activity that more closely mimics physiological conditions. This makes the assay very well suited for low-activity ATPases/kinases.
• Sensitive: The assay is sensitive to low concentrations of ADP, thus requiring less enzyme than other assays; cost savings.
• Universal: The assay can be used with virtually with any ADP-producing enzyme—enables researchers to screen a wider range of enzymes using a single platform.
• Accommodate Wide Range of ATP Levels: The assay can be used at ATP concentrations up to 5mM, important for enzymes with high K_m values for ATP and for mode of action studies.
• Accurate: Accurately measures ADP levels at a wide range of starting ATP concentrations; users assured that activity measured truly reflects enzyme activity and produces accurate IC₅₀s comparable to radioactivity-based assays.

Applications

• Screen library compounds for effects on target enzymes.
• Perform mode of action studies on enzymes of interest.
• Measure activity of immunoprecipitated ATPases/kinase.

ADP-Glo™ Applications Database.

Notes

The ADP-Glo™ Max Assays contain enough reagent to perform the indicated number of reactions in 384-well format using 5μl, 5μl and 10μl of an enzyme reaction, ADP-Glo™ Reagent and ADP-Glo™ Max Detection Reagent, respectively, per sample.

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