ADP-Glo™ Kinase Assay

What does the ADP-Glo™ Assay measure—ADP produced or ATP remaining?

The ADP-Glo™ Assay measures the ADP produced by a kinase reaction, not the ATP that remains. After the kinase reaction, the ADP-Glo™ Reagent depletes leftover ATP, then the Kinase Detection Reagent converts the ADP back into ATP and generates a luminescent signal—so higher kinase activity produces more ADP and more light.

Does the ADP-Glo™ Assay work for all kinases?

The assay is designed to work with virtually any purified kinase, including serine/threonine, tyrosine and lipid kinases, because it detects ADP rather than a kinase-specific substrate modification. Promega also offers Kinase Enzyme Systems covering over 360 individual kinases and Lipid Kinase Assay Reagents for phosphoinositide kinases, built on the same ADP-Glo™ chemistry.

What is the Kinase-Glo® assay principle, and how does it differ from the ADP-Glo™ Assay?

The Kinase-Glo® Assay measures the ATP that remains after a kinase reaction: as kinase activity consumes ATP, luminescent signal falls, so inhibitors cause the signal to rise. The ADP-Glo™ Assay instead measures the ADP produced by the reaction, so its signal falls when kinase activity is inhibited—the opposite direction, using the same luciferase chemistry.

Can ADP-Glo™ be automated for 384- or 1536-well HTS?

Yes. The ADP-Glo™ Assay uses a homogeneous, no-wash biochemical reagent with a stable, non-decaying luminescent signal, so plates can be processed in batches without strictly timed reads—ideally suited to 384- and 1536-well HTS automation. See the Automation of ADP-Glo™ Universal Kinase Assay poster for a validated HTS workflow.

What causes false positives in luminescent kinase assays?

The most common cause is a compound interfering with the luciferase/luciferin detection chemistry itself rather than the kinase—for example, a compound that inhibits Ultra-Glo™ Luciferase can look like a kinase inhibitor. Running a luciferase-interference control, or confirming hits with an orthogonal assay format, helps distinguish true kinase inhibition from detection artifacts.

How do kinase selectivity profiling panels differ from biochemical activity assays?

Activity-based assays, like ADP-Glo™-based Kinase Enzyme Systems, directly measure catalytic turnover. Binding-based panels, such as Eurofins Discovery’s KINOMEscan® platform, measure compound-kinase binding affinity without ATP and don’t reflect catalytic activity. Radiometric activity panels, like Reaction Biology’s HotSpot™ assay, directly measure phosphate transfer across large kinome panels. Each answers a different question.

Why does detecting the reaction product (ADP) improve sensitivity compared to detecting substrate depletion (ATP)?

When kinase activity is low, only a small fraction of ATP converts to ADP—a small dip in a large ATP pool is hard to measure precisely. Detecting the ADP formed instead measures a small quantity against a near-zero background, giving ADP-Glo™ a stronger signal-to-noise ratio for low-activity kinases and low-conversion conditions.

How does ATP concentration affect ADP-Glo™ versus Kinase-Glo® Assay results?

The ADP-Glo™ Assay tolerates ATP concentrations up to 1mM (up to 5mM for ADP-Glo™ Max Assay), which matters for kinases with high Km values that need higher substrate concentrations to reach saturating conditions. The Kinase-Glo® Assay, which measures ATP depletion directly, becomes less sensitive at high starting ATP concentrations, since the fractional change is harder to resolve against a large background. The Kinase-Glo® Max Assay is linear up to 500µM ATP, making it well suited for use with kinases with high Km for ATP, as well as for screening for kinase inhibitors that do not compete at the ATP binding site.

What’s the difference between ADP-Glo™ Kinase Assay and ADP-Glo™ Max Assay?

Both assays use the same two-step ADP-detection chemistry. ADP-Glo™ Max Assay tolerates ATP concentrations up to 5mM (versus 1mM for the Kinase Assay) and is positioned for ATPases as well as kinases, including low-activity ATPases such as membrane transporters and helicases. Choose ADP-Glo™ Max when your target needs higher ATP or isn’t a kinase.

Which assay should I use for primary screening versus kinome-wide selectivity profiling?

For primary screening or mechanism-of-action work on kinases you already have in hand, ADP-Glo™ or Kinase-Glo® biochemical assays are fast and cost-effective. For kinome-wide selectivity profiling across hundreds of kinases using cell-based NanoBRET® Target Engagement technology, consider our Tailored R&D Solutions compound profiling and screening services.