Enabling Breakthrough Discoveries

Scientists today must deliver breakthroughs in technically challenging fields with flat budgets. Our assay technology is designed to help you do more with less. Built on innovation and rooted in sound research methods developed by the Imperiali Laboratory at MIT, our technologies are patented and exclusively licensed to AssayQuant. We leverage our extensive assay development expertise to offer robust products that enable breakthrough discoveries and increase productivity that will improve human health.

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Our product portfolio using the Sulfonamido-oxine (Sox) fluorophore for detection of protein kinase and phosphatase activity is commercialized under the brand name PhosphoSens. Sox was designed to be small and minimally hydrophobic and is well-tolerated across diverse sequences and targets.

  • PhosphoSens: For purified enzymes. Kinetic or endpoint. Excitation Max 360 nm & Emission Max 485 nm, using fluorescence intensity.
  • PhosphoSens-Lysate: Works the same as PhosphoSens but designed for crude cell or tissue lysates.
  • PhosphoSens-Red: Endpoint. Excitation Max 360 nm & Emission Max 620 nm, using Europium (Eu3+), time-resolved fluorescence (TRF) and a 100 μs delay to eliminate interference due to compound autofluorescence.

How It Works

By harnessing chelation-enhanced fluorescence (ChEF) with improved sulfonamido-oxine (Sox) chromophore technology for peptide or protein substrates, we have created a simple yet powerful method to measure the activity of protein kinases or phosphatases using a direct and homogeneous format that provides more information with less effort and accommodates any ATP concentration or sample type. The continuous (kinetic) format is ideal for elucidating drug mechanism of action, potency and profiling, while the Endpoint/Red format is ideal for HTS and SAR. One can switch between formats with the same sensor, to provide seamless coverage across the entire discovery and drug development process.

Why It's Better

Most of the 30-plus commercially available kinase assay platforms use antibodies, coupled reactions, indirect measurements, radioactivity or complex instrumentation for detection. These and other limitations have slowed progress and prevented scientific discovery. Our recent advancements in continuous protein kinase or phosphatase activity assays accelerate discovery by providing better information with reduced effort.

Better technology doesn't have to be more expensive. We understand the need to deliver a solution that is faster, better and cost-effective. Our exclusive PhosphoSens technology is unmatched in terms of simplicity, speed, cost and quality of information.

PhosphoSens Advantages

  • Proprietary CSox-based optimized peptide substrate sensors derived from known physiological sequences
  • Simple add & read homogeneous, direct, and continuous (kinetic) format that uses any ATP concentration and where the level of fluorescence is directly proportional to the amount of phosphorylated product
  • More information with less effort, with quantitative results for potency that align with cell-based assays that accelerate the drug development process and improve efficacy
  • Highly reliable - high accuracy & precision (Z’ > 0.7) and excellent sensor lot-to-lot consistency
  • Runs on standard fluorescence plate readers and is compatible with 96, 384 and 1536-well plate formats
  • Eliminates the use of radioactive or hazardous materials and the requirement for antibodies

PhosphoSens Sensors

AssayQuant has created a robust, high-throughput process for generating superior and fit-for-purpose Sox-based substrates starting with a physiologically relevant or known substrate. Our approach integrates the improved CSox technology from the Imperiali laboratory that allows flanking sequence on either side of the Sox chromophore. These optimized CSox substrates can be used in kinetic or endpoint assays. We have designed both highly generic and highly selective sensors.

  • Generic Sensors – For use with purified enzymes, including recombinant and immunoprecipitation-based assays. For example, the CSox-based substrates AQT0025, 26, 101, 102 & 104 provide very strong signals with over 80 Tyrosine Kinases (TK's) each (both Receptor & Soluble TK forms), providing flexibility, a streamlined workflow and better pricing.
  • Selective Sensors - The same robust process for creating highly generic CSox-based substrates has been applied to maximize selectivity for the target enzyme, allowing activity to be measured in crude cell or tissue lysates. These selective substrates are used with the PhosphoSens-Lysate format.

Performance and Versatility to Meet Your Needs

The PhosphoSens platform delivers flexibility with assay components (use a wide range of ATP, substrate, activators or inhibitors), assay volume, plate configurations (96, 384 or 1536-well), microplate readers and sample types (purified enzyme or crude lysate) – all with a simple to use (add & read) and highly quantitative format that we offer in 3 variations:

  • PhosphoSens: Used with either Generic or Selective Sensors for measuring activity of purified enzymes including recombinant kinases or phosphatases and immunoprecipitation-based enzyme assays. Kinetic or endpoint detection with Excitation Max 360 nm and Emission Max 485 nm.
  • PhosphoSens-Lysate: Used with Selective Sensors optimized for measuring the activity of enzymes in crude cell or tissue lysates that may contain many kinases or phosphatases. Kinetic or endpoint detection with Excitation Max 360 nm and Emission Max 485 nm.
  • PhosphoSens-Red: Use with either Generic or Selective Sensors with the appropriate sample type. Endpoint detection with Excitation Max 360 nm and Emission Max 620 nm, where the much higher emission wavelength and time-resolved fluorescence eliminates interference from compound autofluorescence.

The kinetic format captures the complete progress curve (fluorescence intensity over time) for every condition and with no additional handling. The endpoint format can use a stop solution to allow processing of plates in batch mode to provide higher-throughput.

Current target enzyme classes include:

  • Protein Kinases - Use optimized CSox-based peptide substrate sensors, where phosphorylation of the Ser, Thr or Tyr residue results in an immediate increase in fluorescence as a direct measure of enzyme activity.
  • Protein Phosphatases - Use optimized CSox-based phosphopeptide substrate sensors, where dephosphorylation of the pSer, pThr or pTyr residue results in an immediate decrease in fluorescence as a direct measure of enzyme activity.
  • Diacylglycerol (Lipid) Kinases (DGKs) - Use an optimized Sox derivative as the sensor, where phosphorylation of different diacylglycerol (DAG) substrates to create phosphatidic acid (PA) results in an immediate increase in fluorescence that is a direct measure of DGK enzyme activity performed. This continuous assay is performed using a lipid-detergent micelle format.