PhosphoSens® Technology
A Direct, Continuous Fluorescence System for Measuring Enzyme Activity and Inhibition
Developed by MIT scientists, the PhosphoSens® platform is a proprietary assay system for measuring kinase and phosphatase activity in real time. Built on our innovative Sox-based Sensor Peptide Technology, this platform enables direct, continuous, and quantitative measurement of phosphorylation, delivering richer insights, faster workflows, and confident decision-making from discovery through lead optimization.
Direct Enzyme Activity Detection Powered by Technology-Packed Sensor Peptide Substrates
Most phosphorylation assays rely on indirect detection, secondary reagents, or endpoint readouts. PhosphoSens® assays are different — they measure enzyme activity directly by monitoring substrate phosphorylation in real time. Here's how:
Sensor Peptide Substrates:
Each peptide substrate is derived from an extensive library of >50,000 sequences and further optimized for target specific assay performance.
Covalently attached in close proximity to the Serine, Threonine, or Tyrosine phosphorylation site (± 2-5 residues) is the readout molecule: Sulfonamido-oxine (Sox).
Sox Fluorophore:
In the absence of phosphorylation, the Sox shows low affinity for Mg(II); upon phosphorylation, Mg(II) affinity is enhanced due to the advantageous chelate effect, involving the Sox and the introduced phosphate group, and fluorescence is turned on.
Chelation-Enhanced Fluorescence (ChEF):
The Sox Fluorophore reports peptide phosphorylation by the target kinase via chelation-enhanced fluorescence (ChEF). The fluorescence-sensing mechanism is based on the chelate effect introduced by phosphorylation of the Ser/Thr/Tyr residue proximal to CSox, which enhances magnesium (Mg²⁺) binding affinity compared to the unphosphorylated species, and results in increased fluorescence emission intensity (2-10-fold).
As the enzyme acts on the substrate, phosphorylation induces a chelation‑enhanced fluorescence (ChEF) signal proportional to phosphorylation level.
One Platform. Multiple Applications.
PhosphoSens Technology is designed to meet the full spectrum of your assay needs — from purified enzyme studies to complex biological samples — and is available in two flexible assay formats. By customizing substrate sequences and assay conditions, we can tailor the platform to meet the specific needs of different experimental contexts:
Assay Formats
Continuous, Kinetic Fluorescent Intensity (FI) Assays
Capture real-time, kinetic data throughout the entire enzyme reaction at the substrate level, where the level of fluorescence is directly proportional to the amount of phosphorylated product.
Endpoint Time-Resolved Fluorescence (TRF) Assays
Designed for high thoughput screening applications, capture a single fluorescence measurement without signal contamination, utilizing the same reaction conditions and set up as our Kinetic assays.
Sample Types:
Active Recombinant Proteins
Gain deep mechanistic insights from recombinant kinase or phosphatase reactions in clean, defined systems.
Cell Lysates & Tissue Homogenates
Directly measure endogenous kinase activity in biologically relevant and complex sample matrices, delivering meaningful data for pathway analysis and disease relevance.
Advantages
Continuous
Kinetic progress curves in real time — no assumptions, no extrapolation. Greater accuracy and mechanistic insight.
Direct
No coupling enzymes or antibodies. Activity is measured directly from the substrate level.
Physiologically Relevant
Substrates designed from native sequences and assays run under true physiological ATP levels (μM–mM).
Streamlined Workflow
Add & read—no washes, immobilization, or custom equipment needed. Ideal for HTS and automation.
Simple workflow for continuous and red-shift formats
Identify and Validate the Right Candidates Faster with Richer Data
Applications:
- Kinome Profiling: Gain detailed kinetic insights into compound selectivity and mechanisms of action (MOA) by profiling against a comprehensive panel of over 400 wild-type kinases. Experiments can be conducted under physiological conditions with high or low ATP levels to match your research needs.
- IC₅₀ or Kᵢ Determination: Evaluate compound potency using 12-point dose-response curves. Flexible assay conditions include the presence of BSA or DTT, high or low ATP levels, and the ability to analyze protein complexes or crude lysates.
- kinact/Kᵢ for Time-Dependent Inhibition: Quantify covalent compound inhibition dynamics with 24-point time-dependent assays. These provide precise measurements of inactivation rates (kinact) and covalent binding affinities (Kᵢ).
- Residence Time: Characterize compound binding kinetics using the Jump Dilution method, with a detection range from 30 seconds to 48 hours. This service provides valuable data on compound dissociation rates for informed drug discovery.
- Mechanism of Action (MOA): Uncover how your compound interacts with kinases through detailed studies using ATP, peptide, or lipid substrates. Tailored to your target and research goals, MOA assays provide actionable insights.
- EC50: Assess compound potency for target activation with EC₅₀ measurements, delivering reliable data on dose-dependent activation to guide lead optimization.
Indications:
- Cancer: Exploring kinase-driven pathways, such as those involved in uncontrolled cell proliferation and survival, and metastasis.
- Neurological Disorders: Investigating signaling pathways regulated by kinases or phosphatases including Parkinson’s Disease, stroke, and various tauopathies such as Alzheimer's disease (AD), frontotemporal dementia, corticobasal degeneration (CBD), and chronic traumatic encephalopathy, characterized pathologically by the deposition in the brain of aggregates of the insoluble hyperphosphorylated form of the tau protein (p-tau).
- Cardiovascular Diseases: Studying kinases or phosphatases that affect heart function, blood vessel regulation, cardiac hypertrophy, myocardial infarction and hypertension.
- Immunology: Evaluating kinases or phosphatases involved in immune cell activation, chronic inflammation, and autoimmune diseases where signaling is deregulated.
- Metabolic Disorders: Characterize kinases or phosphatases implicated in diabetes, insulin resistance, obesity, dyslipidemia, and metabolic dysfunction-associated fatty liver disease (MAFLD).
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