In Vitro Protein Kinase Assays

Q: What is the difference between radiometric and non-radiometric kinase assays?

Radiometric assays use [γ-32P]- or [γ-33P]-ATP to directly measure phosphate incorporation, offering the highest sensitivity and serving as the biochemical gold standard. Non-radiometric assays (fluorescence, luminescence, mobility shift, ELISA) eliminate radioactivity hazards, reduce disposal costs, and are generally preferred for high-throughput screening. The choice depends on sensitivity requirements, throughput, and safety constraints.

Q: How is the ATP concentration selected for IC50 determination?

For ATP-competitive inhibitors, we typically perform IC50 determinations at an ATP concentration equal to the Km of the kinase for ATP. Under these conditions, IC50 = 2 × Ki (for competitive inhibitors), allowing direct conversion to the enzyme-specific inhibition constant using the Cheng–Prusoff equation. This standardization ensures comparability across different kinases and laboratories.

Q: How long does a typical kinase assay project take?

Standard single-target IC50 determinations can be completed within 2–3 weeks from compound receipt. Kinome profiling and custom assay development projects require 4–8 weeks depending on scope and complexity. Expedited timelines are available upon request.

Protein kinases are enzymes that catalyze the phosphorylation of substrate proteins by transferring the γ-phosphate group from ATP to the hydroxyl groups of serine, threonine, or tyrosine residues. This post-translational modification alters substrate conformation, activity, and protein–protein interactions, thereby regulating essential cellular processes including signal transduction, cell cycle progression, metabolism, and gene expression. The human kinome comprises more than 500 protein kinases, broadly classified into receptor tyrosine kinases, non-receptor tyrosine kinases, serine/threonine kinases, and cyclin-dependent kinases (CDKs). Dysregulated kinase activity is implicated in numerous pathological conditions, most notably cancer, making kinases one of the most extensively pursued target classes in drug discovery. Profacgen provides comprehensive in vitro protein kinase assay services that deliver quantitative, reproducible data to support kinase inhibitor screening, mechanistic studies, and lead compound optimization.

In vitro protein kinase assay formats and detection methods

What We Offer: In Vitro Kinase Assay Services

Profacgen offers a flexible, high-throughput in vitro kinase assay platform designed to accommodate diverse project requirements—from single-target IC₅₀ determination to broad kinome profiling. All assays are performed in 96- or 384-well microplate formats, ensuring low reagent consumption, rapid turnaround, and compatibility with high-throughput screening (HTS) workflows. Our experienced team works closely with clients to select the optimal assay format, kinase construct, and substrate based on the biological question and downstream application goals.

Assay Formats

We support multiple detection modalities, allowing format selection to be tailored to sensitivity requirements, throughput needs, and safety considerations:

Radiometric assays

Using [γ-³²P]- or [γ-³³P]-labeled ATP, these assays directly measure phosphate incorporation into the substrate. Radiometric methods remain the gold standard for sensitivity and accuracy, particularly for validating novel kinase–substrate pairs.

Fluorescence-based assays

Includes fluorescence polarization (FP), fluorescence resonance energy transfer (FRET), and time-resolved FRET (TR-FRET). These homogeneous formats enable real-time monitoring with high signal-to-noise ratios and are well suited for miniaturized screening.

Luminescence-based assays

ATP-depletion formats quantify kinase activity by detecting ADP formation or ATP consumption via coupled luciferase reactions. These non-radioactive methods offer excellent dynamic range and are ideal for large-scale inhibitor libraries.

Mobility shift assays

Capillary electrophoresis–based separation distinguishes phosphorylated from non-phosphorylated peptide substrates based on charge-to-mass ratio, providing direct, quantitative readouts without antibodies or radioactivity.

ELISA-based assays

Sandwich or competitive immunoassays using phospho-specific antibodies capture and quantify phosphorylated substrates, offering high specificity for defined phosphorylation sites.

Supported Kinase Targets

Our assay portfolio covers a broad spectrum of the human kinome, including but not limited to:

Receptor tyrosine kinases (RTKs): EGFR, HER2, VEGFR, PDGFR, FGFR, MET, RET
Non-receptor tyrosine kinases: Src family, Abl, Jak family, Syk, BTK
Serine/threonine kinases: AKT/PKB, mTOR, Raf, MEK, ERK, GSK-3β
Cyclin-dependent kinases (CDKs): CDK1, CDK2, CDK4/6, CDK7, CDK9
Other kinases: CK1, CK2, PI3K family, AMPK, LKB1, and custom targets upon request

Both wild-type and disease-relevant mutant constructs are available. Custom kinase expression and purification can be arranged for targets not currently in our inventory.

Assay Principle and Workflow

The fundamental principle of an in vitro kinase assay is the detection of phosphoryl transfer from ATP to a substrate. Profacgen follows a standardized workflow to ensure data quality, reproducibility, and regulatory compliance:

In vitro protein kinase assay workflow

Assay Development & Optimization: For each kinase–substrate pair, we determine optimal enzyme concentration, ATP K_m, reaction time, and buffer conditions under initial velocity constraints. This step ensures linear kinetics and robust signal-to-background ratios.
Compound Preparation: Test compounds are prepared as DMSO stock solutions and serially diluted across a concentration range (typically 10-point, half-log dilutions) in assay buffer.
Reaction Setup: In a microplate format, kinase, substrate, and test compound are combined and pre-incubated to allow compound binding. The reaction is initiated by addition of ATP at a defined concentration (often at or near the K_m for ATP-competitive inhibitor studies).
Incubation: Reactions proceed at 30 °C for a predetermined duration within the linear phase of product formation.
Detection: The reaction is terminated and the signal generated according to the selected assay format—radiometric counting, fluorescence/luminescence plate reading, electrophoretic separation, or antibody-based capture.
Data Analysis: Raw signals are normalized to positive (no inhibitor) and negative (no enzyme) controls. Percent inhibition is calculated and fitted to a four-parameter logistic model to derive IC₅₀ values. For ATP competition studies, IC₅₀ values are determined across multiple ATP concentrations and analyzed using the Cheng–Prusoff equation to calculate K_i.

Service Capabilities

Capability	Description	Typical Deliverable
Single-Dose Screening	Primary screening of compound libraries at a single concentration (e.g., 1 µM or 10 µM) against one or multiple kinases to identify hits.	Percent inhibition table; selectivity score
IC₅₀ Determination	Full dose–response curves (10-point, duplicate or triplicate) for confirmed hits to quantify inhibitor potency.	IC₅₀ value; Hill slope; R² fit statistic
ATP Competition Studies	IC₅₀ determination at varying ATP concentrations to classify inhibitors as ATP-competitive (Type I/II) or allosteric (Type III).	K_i (competitive); mode-of-action classification
Kinase Selectivity Profiling	Panel screening against 10–410 kinases to assess off-target activity and polypharmacology.	Selectivity matrix; heat map; SAR summary
Mechanism-of-Action (MoA) Studies	Time-dependent inhibition, reversibility assays, and substrate competition to elucidate binding kinetics.	K_on/K_off; reversibility classification
Custom Assay Development	Development and validation of novel assays for non-standard kinases, substrates, or detection requirements.	Qualified assay protocol; validation report

Applications

In vitro kinase assays are indispensable across the drug discovery and development pipeline:

Kinase inhibitor screening: Primary and secondary screening of small-molecule libraries, natural product extracts, and fragment collections to identify inhibitors with desirable potency and selectivity profiles.
IC₅₀ determination: Quantitative ranking of lead compounds to guide structure–activity relationship (SAR) studies and prioritize candidates for advancement.
ATP competition studies: Differentiation of ATP-competitive inhibitors from allosteric modulators, informing medicinal chemistry strategies and intellectual property positioning.
Hit validation and lead optimization: Confirmation of on-target activity, assessment of selectivity against closely related kinases, and elimination of assay artifacts or promiscuous inhibitors.
Biochemical mechanism studies: Elucidation of inhibitor binding mode, reversibility, and residence time to predict cellular efficacy and off-rate liabilities.

Service Advantages

Comprehensive Kinase Coverage: Broad panel of purified kinases spanning major therapeutic target classes
Multiple Assay Formats: Radiometric, fluorescence, luminescence, mobility shift, and ELISA-based options to match project needs
Standardized Workflow: Rigorous assay development under initial velocity conditions ensures reproducible, comparable IC₅₀ and K_i values
High-Throughput Compatibility: 384-well microplate formats with low reagent consumption for efficient screening
Experienced Scientific Team: Deep expertise in enzyme kinetics, inhibitor characterization, and assay troubleshooting
Integrated Data Analysis: Automated curve fitting, statistical analysis, and structured reporting to accelerate decision-making

Deliverables

Upon project completion, clients receive a comprehensive data package including:

Raw data: Unprocessed plate reader outputs, radiometric counts, or electropherograms in Excel or CSV format
Dose–response curves: Graphical plots with fitted four-parameter logistic curves, including 95% confidence intervals
IC₅₀ / K_i values: Tabulated potency parameters with replicate statistics and assay conditions
Study report: Detailed protocol description, quality control metrics, and interpretive summary prepared by our scientific team

Frequently Asked Questions (FAQs)

Q: What is the difference between radiometric and non-radiometric kinase assays?

A: Radiometric assays use [γ-³²P]- or [γ-³³P]-ATP to directly measure phosphate incorporation, offering the highest sensitivity and serving as the biochemical gold standard. Non-radiometric assays (fluorescence, luminescence, mobility shift, ELISA) eliminate radioactivity hazards, reduce disposal costs, and are generally preferred for high-throughput screening. The choice depends on sensitivity requirements, throughput, and safety constraints.

Q: How is the ATP concentration selected for IC₅₀ determination?

A: For ATP-competitive inhibitors, we typically perform IC₅₀ determinations at an ATP concentration equal to the K_m of the kinase for ATP. Under these conditions, IC₅₀ = 2 × K_i (for competitive inhibitors), allowing direct conversion to the enzyme-specific inhibition constant using the Cheng–Prusoff equation. This standardization ensures comparability across different kinases and laboratories.

Q: Can you profile compounds against the entire human kinome?

A: Yes. Profacgen offers kinome-wide profiling against panels of up to 410 kinases, enabling comprehensive selectivity assessment. Primary screens are typically performed at a single compound concentration (e.g., 1 µM), followed by full IC₅₀ curve determination for kinases showing significant inhibition (>50%).

Q: What substrates are used in the assays?

A: We use validated peptide substrates, recombinant protein substrates, or physiologically relevant full-length proteins depending on the kinase and assay format. For novel kinases without established substrates, we can assist in substrate identification and assay development.

Q: How long does a typical kinase assay project take?

A: Standard single-target IC₅₀ determinations can be completed within 2–3 weeks from compound receipt. Kinome profiling and custom assay development projects require 4–8 weeks depending on scope and complexity. Expedited timelines are available upon request.

Q: Do you provide assay validation for regulatory submissions?

A: While our standard biochemical assays are designed for drug discovery research, we can adapt protocols and documentation to support IND-enabling or regulatory submission requirements. Please contact our team to discuss specific compliance needs.

References:

Qing T, Liu J, Liu F, Mitchell DC, Beresis RT, Gordan JD. Methods to assess small molecule allosteric modulators of the STRAD pseudokinase. In: Methods in Enzymology. Vol 667. Elsevier; 2022:427-453. doi:10.1016/bs.mie.2022.03.041