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Kinase Panel Screening

Biochemical Kinase Assays

Selectivity profiling, screening, and IC50 determination on the industry’s largest kinase activity platform

Direct radiometric activity assays (HotSpot and 33PanQinase) plus luminescent ADP-Glo for lipid kinases. 870+ kinases, native protein and peptide substrates, physiological ATP, delivered from US and EU facilities.

Profiling Selectivity Across the Human Kinome

The human kinome contains more than 500 protein kinases and 20 more lipid kinases that share a conserved ATP-binding catalytic core. That structural homology makes selectivity the central challenge of kinase drug discovery: a compound optimized against one target frequently inhibits others, and off-target kinase inhibition is a leading driver of toxicity and late-stage attrition. Quantitative selectivity profiling, measured directly as inhibition of catalytic activity, rather than inferred from binding, gives medicinal chemistry an accurate parameter to optimize against and surfaces liabilities before they reach the clinic.

How Kinase Profiling Works

  • Your compound is tested against a defined set of kinases, from a single target to the full kinome, at single-dose or multi-point IC50 format.
  • Activity of protein kinases are measured by direct radiometric transfer of 33P-phosphate to substrate; activity of lipid kinases are measured by ADP-Glo.
  • Results are returned as percent inhibition, activity, or IC50, with raw data and curve fitting in Prism.
  • Selectivity is visualized on the interactive Kinase Mapper across the kinome tree.

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Our Services

Kinase Selectivity Profiling

Screen one subfamily or the entire human kinome with a large collection of predefined panels plus free-choice selections. IC50-based profiling delivers a quantitative selectivity parameter for medicinal chemistry optimization and off-target assessment, visualized on the Kinase Mapper.

Single-Target Activity and IC50

Hit confirmation, dose-response, and mechanism studies on individual kinases. Profile wild-type and clinically relevant mutants side by side, and resolve ATP-competitive, substrate-competitive, covalent, and allosteric modes of inhibition.

Library and Fragment Screening

High-throughput primary screening of your library or ours, from diverse small molecules to focused, FDA-approved, and covalent fragment collections. Cherry-picking, compound management, and IC50 follow-up are available end to end.

Custom Assay Development

Targets outside the standard library are built to order: plasmid construction, expression, purification, substrate identification, and assay optimization for specialized requirements, such as time dependent inhibition, covalent binding, or inhibition of inactive forms. Native peptide and full-length protein substrates are supported without modification.

Detection Technologies

HotSpot (Malvern, US) 33PanQinase (Freiburg, DE) ADP-Glo (both sites)
Detection principle Radiometric 33P, filter-membrane capture Radiometric 33P, scintillant ScintiPlate capture Luminescent ADP detection (Promega)
What it measures Phosphorylated substrate, direct Phosphorylated substrate, direct ADP formation, indirect measure of phosphorylation
Substrates Native peptide and full-length protein Native peptide and full-length protein Primarily lipid kinase substrates
ATP conditions 1 µM, 10 µM, apparent ATP-Km to 100 µM or 1 mM physiological conditions Apparent ATP-Km Up to 1 mM
Kinetics Time-course reads available Endpoint Endpoint
Library ~830 assays, deepest mutant and fusion coverage ~517 assays, CDK/cyclin complexes and fragment constructs 23+ lipid kinases

Biochemical Kinase Assays Explained

HotSpotTM from Reaction Biology is the gold-standard radiometric assay and default choice for most kinase programs. It measures the catalytic event of phosphorylation directly, transferring 33P-phosphate from ATP onto substrate and capturing the phosphorylated product on a filter membrane at our US facility. The assay protocol for HotSpot is available here.

33PanQinaseTM applies the same direct radiometric chemistry at our European facility, capturing product on scintillant-coated ScintiPlates, and adds complementary coverage including CDK/cyclin complexes. The assay protocol for 33PanQinase is available here.

ADP-Glo is a complementary luminescent format that measures the ADP byproduct of the kinase reaction and is the required format for lipid kinases.

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Platform Advantages

Direct Activity Measurement

We measure phosphate transferred to substrate, the catalytic event itself, not binding, ADP, or FRET proxies. Binding does not equal inhibition, and proxy readouts cannot distinguish active-site engagement from non-functional binding.

The Largest Kinase Library

870+ kinase assays including 260+ clinically relevant mutants and 32 oncogenic fusion proteins, the broadest commercially available activity-based panel.

Native, Unmodified Substrates

Full-length proteins and native peptides are accepted without labeling or chemical modification, unlike platforms restricted to modified Sox-peptides, peptide-only, or FRET-labeled substrates.

Physiological ATP Without Compromise

Radiometric detection is unaffected by high ATP, so assays run at physiological 1 mM ATP without the signal degradation seen on luminescent and fluorescent platforms. Available for 340 wild-type kinases in HotSpot format.

Slow-Binding and Covalent Inhibitors

Compound can be pre-incubated with kinase before ATP and substrate are added, up to about two hours on request, to detect slow-binding and time-dependent inhibitors.

Dual-Site US and EU Delivery

The only kinase CRO running radiometric activity assays at both a US and a European facility. EU clients avoid trans-Atlantic compound shipping, and the two sites carry complementary specializations.

High-Throughput Automation

Echo acoustic dispensing and automated liquid handling support primary screening at scale across the library.

Predictable Turnaround

Typical panels and projects completed within 10-15 business days. Single-target and custom work is reported in 10 business days, regardless of compound receipt date. Expedited timelines are available.

Assay Setup and Requirements

Selectivity, Mechanism, and Reproducibility

  • Why profile with IC50 values
  • Mechanism of action via SAR
  • Reproducibility: BCR-ABL inhibitor (33PanQinase)
  • Reproducibility: Staurosporine control (HotSpot)
  • Selectivity profiling: FLT3 inhibitor (33PanQinase)
Why profile with IC50 values

Protein kinases share a conserved catalytic core and high structural and sequence similarity, which limits inhibitor selectivity and can produce off-target toxicity that ends otherwise promising candidates. IC50-based profiling quantifies how potency differs across the kinome, enabling a precise judgment of selectivity. It is designed to:

1. Provide an accurate guiding parameter for optimizing kinase selectivity during medicinal chemistry
2. Confirm that observed functional effects are driven by the intended target and not off-target kinases, and surface potential adverse effects early
3. Provide an accurate comparison against a reference compound

Mechanism of action via SAR

Structure-activity relationship analysis with IC50 potency assays elucidates how a shortlisted inhibitor engages its target. By comparing IC50 values across the wild-type kinase and variants carrying targeted domain mutations, we resolve the critical binding interactions and classify the inhibitor as covalent, ATP-competitive, or substrate-competitive. This confirms the activity profile and directs targeted optimization.

Reproducibility: BCR-ABL inhibitor (33PanQinase)

IC50 values of a BCR-ABL kinase inhibitor were determined for 320 wild-type kinases across two independent experiments and compared. The high R-squared value shows strong correlation between runs. IC50 was determined with 6 compound concentrations in singlicate using the 33PanQinase assay.

Reproducibility: Staurosporine control (HotSpot)

The control compound staurosporine was tested over 6 months across 12 independent experiments. IC50 was determined with 10 compound concentrations in singlicate using the HotSpot assay, demonstrating long-term assay stability.

Selectivity profiling: FLT3 inhibitor (33PanQinase)

IC50 values for an FLT3 inhibitor were determined across 320 wild-type protein kinases, with the lowest-IC50 targets named. Profiling at this scale distinguishes potency differences across the kinome and supports a precise selectivity judgment.

Frequently asked questions

What is kinase selectivity profiling and why does it matter?

It is the testing of a compound against many kinases to quantify how selectively it inhibits its intended target. Because kinases share a conserved ATP-binding site, off-target inhibition is common and is a leading cause of toxicity and attrition, so selectivity data directly informs candidate selection and optimization.

What is the difference between HotSpot and 33PanQinase?

Both are radiometric activity assays that measure phosphorylation directly using 33P. HotSpot, run in the US, captures phosphorylated substrate on a filter membrane. 33PanQinase, run in Germany, uses scintillant-coated ScintiPlates. They differ in capture format, assay conditions, and site, and they carry complementary target coverage.

Why radiometric detection instead of fluorescent or luminescent?

Radiometric assays measure the catalytic event of phosphorylation directly, so they are not subject to the optical interference and signal degradation that affect coupled luminescent and fluorescent formats, and they remain reliable at physiological 1 mM ATP.

Do you measure activity or binding?

Activity. We quantify inhibition of catalytic phosphoryl transfer. Binding-based panels report displacement, which cannot distinguish active-site inhibition from non-functional binding and run without ATP in the reaction.

Can you run assays at physiological ATP?

Yes. Radiometric detection is unaffected by high ATP, and 1 mM ATP is available for 340 wild-type kinases on the HotSpot platform, in addition to standard concentrations of 1 µM, 10 µM, and apparent ATP-Km.

How many kinases and which panels do you offer?

870+ kinase assays, including 260+ clinically relevant mutants, 32 oncogenic fusions, and lipid kinases. Choose a predefined panel, a specialty mutant profiler, or a free-choice selection of any targets in the library.

Can you detect slow-binding or covalent inhibitors?

Yes. Compound can be pre-incubated with the kinase/substate mixture for varying time before ATP addition to reveal slow-binding and time-dependent inhibition. Time-course reads are available on HotSpot assay to further characterize kinetic parameters of slow binding inhibitors

What do I need to send, and how long does it take?

Requirements differ by site. For HotSpot (US) panel profiling, send 100 µl of a 10 mM DMSO stock, or solid material. For 33PanQinase (Germany), send a 100x stock in 100% DMSO: 1900 µl for one concentration in duplicate; 950 µl of each concentration for two concentrations in singlicate; or, for a 6-point IC50, 1100 µl at the highest assay concentration. Solid material is also accepted. Most panels are reported in 10 to 15 business days, and single-target or custom work in 10 business days, regardless of when compound is received.