PhosphoSens-Red Enzyme Screening Assays
High-throughput TRF detection backed by the same substrate chemistry as PhosphoSens-Kinetic.
PhosphoSens-Red assays deliver rapid, TRF-based endpoint measurements optimized for hit identification, triage, and large-scale compound profiling — all while preserving the advantages of direct substrate turnover detection.
Every assay can also be read kinetically, enabling seamless transition from screening to deeper mechanistic characterization.
Assay Principle & Workflow
PhosphoSens assays leverages AssayQuant’s Proprietary Detection Technology to provide real-time, continuous measurements of kinase activity by directly detecting substrate phosphorylation throughout the kinase reaction.
Sensor Peptide Substrates are developed with the Sox readout molecule, covalently attached via a cysteine residue in close proximity to the phosphorylation site (± 2-5 residues).
As the kinase acts on the substrate, phosphorylation induces a chelation‑enhanced fluorescence (ChEF) signal proportional to phosphorylation level.
Add
Active enzyme is added to reaction mix containing a Sensor Peptide Substrate, catalyzing phosphotransfer from ATP to the substrate in real time.
Read
Add plate to reader and monitor kinase activity throughout the reaction. Signal is directly proportional to the phosphorylation level of the substrate.
Stop & Read
Add Europium to displace magnesium, which stops the reaction and shifts the emissions of Sox to ~620 nm. Measure using time-resolved
fluorescence with a 100-150 µsec delay and a 300-500 µsec data acquisition time, thereby
eliminating any interference due to compound autofluorescence.
Time-Resolved Fluorescence (TRF) Enhances Screening Robustness
In Red mode, europium displaces magnesium to stop the reaction and shift the fluorophore’s emission into the long-lived TRF window. This removes short-lived background signals and compound autofluorescence, enabling highly reproducible endpoint measurements ideal for high-throughput screening.
Designed for Efficient, High-Throughput Kinase and Phosphatase Screening
Ideal for Hit Identification & Early Triage
Quickly determine compound activity before moving to full kinetic characterization.
Compatible With 384- and 1536-Well Formats
Scale seamlessly for automated workflows.
Physiologically Relevant Conditions
Maintain biologically meaningful ATP concentrations and cofactors during screening.
Cost-Effective Screening
Reduced read frequency and simplified workflow minimizes reagent use and instrument time.
Dual-Mode Measurement from the Same Assay
Collect both TRF endpoint (Red) and continuous kinetic data from a single reaction, allowing hit confirmation, kinetic insight, and screening readouts without repeating experiments.
PhosphoSens-Red vs PhosphoSens-Kinetic: Which Should You Use?
PhosphoSens-Red is optimized for rapid compound screening, while PhosphoSens-Kinetic provides deeper mechanistic insight. Use the table below to determine the best mode for your experiment.
| Parameter | Kinetic (Full Profiling Mode) | Red (Screening Mode) |
|---|---|---|
| Read Mode | Continuous real-time fluorescence | TRF Stop-and-Read |
| Data Depth | Full progress curves | Single optimized endpoint |
| Ideal Use Case | Mechanistic studies, IC50, SAR | Hit ID, triage, primary screens |
| Supported Throughput | ★★★☆☆ | ★★★★★ |
| Cost per Well | Moderate | Lower |
| ATP Compatibility | Physiological | Physiological |
| Kinetic Parameters | Initial velocity, kobs, kinact/Ki, mechanism | Not derived from Red mode |
| Dual-Mode Capability | Switch seamlessly to full kinetic analysis for hits | Collect TRF endpoint + kinetic data from same well |
Applications Where Red Excels:
-
Primary kinase inhibitor screens
-
Hit triage and confirmation
-
Medium-throughput selectivity profiling
-
Rapid inhibitor ranking
-
Screening assays requiring physiological ATP levels
-
Profiling large panels of mutant kinases
Understand How Our Technology Can Support Your Next Discovery
Deep Kinetic Characterization: When IC50 Isn't Enough
A continuous assay, like our PhosphoSens assay, that measures the catalytic event of phosphorylation in real time enables determination of kinetic parameters that power your drug discovery research.
Continuous vs. Endpoint Kinase Assays: What You Need to Know
PhosphoSens-Red Kinase Inhibitor HTS Protocol
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