Unique Continuous Enzyme & Kinase Inhibitor Activity Assays
A Powerful Approach for Understanding Kinase and Phosphatase Activity
Search Our Product Catalogs of PhosphoSens Sensor Peptide Substrates
PhosphoSens-Kinetic Kinase Assays
PhosphoSens-Red Kinase
Assays
PhosphoSens-Kinetic Phosphatase Assays
Enzyme and Inhibitor Activity Assays For Target Biology, Drug Discovery and Development, and Research
AssayQuant’s patented PhosphoSens® Technology forms the basis of an extensive portfolio of more than 700 enzyme activity assays. Each assay features novel sensor peptide substrates available to purchase in the form of a Discovery Kit or bulk Sensor Peptide Substrate. For additional product information, continue reading below.
One Technology, Multiple Assay Formats That Work Across The Pre-Clinical Drug Development Continuum
PhosphoSens-Kinetic: Fluorescence Intensity (FI) Assays
PhosphoSens continuous assays utilize a simple add-and-read protocol, providing the capability for real-time, kinetic measurements, with a straight-forward, fluorescence intensity (FI) measurement. These assays provide high accuracy and precision by allowing measurement of the initial reaction rate (measured as the slope of the initial linear region), typically during the first 30-60 minutes and include many data points, with readings performed every 30 seconds to two minutes.
This continuous assay format also allows for correction of compound autofluorescence, because this background signal does not change over time and can be corrected for by subtracting background signals (includes compound and all components of the reaction besides the enzyme) from the total fluorescence (all components including enzyme) to determine the net signal at each time point.
The continuous format is compatible with essentially any commercially available fluorescence microplate reader capable of kinetic readings, and associated laboratory automation equipment. No stop solution or quench/develop step is needed, minimizing time, while maximizing throughput.
PhosphoSens-Red: Time-Resolved Fluorescence (TRF) Assays
Because many compound libraries contain auto-fluorescent compounds, AssayQuant offers PhosphoSens Red assays that allow detection at longer (>600 nm) wavelength, with the same excitation maximum, and use time-resolved fluorescence (TRF). We recommend using a 100 µsecond delay between excitation and detection at the emission wavelength, with a 300 µsecond acquisition time, to avoid signal contamination from fluorescent materials in the reaction mixture, which have a very short (nanosecond) lifetime.
Our end-point assays utilize the same reaction set up as our continuous assays, and once the reaction is run, a Europium ion (Eu3+) based reagent is added to each well, which displaces Mg++ in the chelation complex. Excitation of the Sox fluorophore sensitizes the europium luminescence, producing light with a 615 nm (red) maximum that can be measured using standard filters.
The PhosphoSens Red assay can often use the same PhosphoSens substrate as is used for the continuous format. However, some highly acidic substrates (e.g., those often used for tyrosine kinases), can give rise to low signal to background ratios with the red format. To address this situation, we have identified alternative sensor-peptide sequences that allow for an optimized PhosphoSens Red assay, with signal to background ratios of 2 or greater, which provide Z’ values of 0.7 or higher (0.5 is considered excellent). In these cases, the same sequence can be used for the continuous format, with the same sensitivity or only a small decrease in sensitivity of the assay.
The Advantages of PhosphoSens
Continuous
The level of fluorescence is measured throughout the entire enzyme reaction and is directly proportional to the amount of phosphorylated product.
Direct
Enzyme activity is measured at the substrate level, meaning you don't need to worry about false positives and negatives caused by other formats of detection.
Streamlined Workflow
More information with less effort. With a simple add & read workflow, simply initiate the enzyme reaction and add your plate to the reader and capture a progress curve in every well.
Biologically Relevant
Compatible with low to physiological [mM] levels of ATP concentrations, use physiological Mg2+, Mn2+, Ca2+ ion concentrations and peptide sequences derived from physiologically relevant substrates.
Reliable and Accurate
A continuous format yields an actual reaction rate determined from dozens of data points, giving you confidence in your data.
Non-Disruptive
Runs on standard fluorescence microplate readers and is compatible with 96, 384 and 1536-well plate formats.
Bulk Sensor Peptide Substrate
Designed for higher throughput applications, each PhosphoSens Senor Peptide Substrate can be purchased as lyophilized 1mg aliquots.
Each mg is sufficient for ~2,500 assays in a Low Volume 384-well White Flat Bottom Polystyrene NBS Microplate (10 μM Sensor Peptide Substrate with 25 μL final reaction volume).
PhosphoSens-Kinetic Reagent Pack
PhosphoSens-Kinetic Reagent Packs are designed to pair with 1 mg of any Sensor Peptide Substrate and include sufficient volumes of required reagents to perform 3,000+ reactions in a low‐volume 384‐well plate (10 μM sensor peptide in 25 μL reaction volume).
| COMPONENT | DESCRIPTION | VOLUME | ||
| ATP Solution, 100 mM | 100 mM ATP in nuclease-free water | 1.5 mL | ||
| DTT Solution, 1 M | 1 M DTT in nuclease-free water | 0.15 mL | ||
| Enzyme Reaction Buffer, 10X | 500 mM HEPES, pH 7.5, 0.1% Brij-35, 100 mM MgCl2 | 15 mL | ||
| Enzyme Dilution Buffer, 5X | 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, 1 mg/mL BSA | 35 mL | ||
| EGTA Solution, 550 mM | 550 mM EGTA in 2 M NaOH | 0.15 mL | ||
PhosphoSens-Red Reagent Pack
PhosphoSens-Red Reagent Packs are designed to pair with 1 mg of any Sensor Peptide and have sufficient volumes of required reagents to perform 3,000+ reactions in a low‐volume 384‐well plate (10 μM sensor peptide in 25 μL reaction volume).
| COMPONENT | DESCRIPTION | VOLUME | ||
| ATP Solution, 100 mM | 100 mM ATP in nuclease-free water | 1.5 mL | ||
| DTT Solution, 1 M | 1 M DTT in nuclease-free water | 0.15 mL | ||
| Enzyme Reaction Buffer, 10X | 500 mM HEPES, pH 7.5, 0.1% Brij-35, 100 mM MgCl2 | 15 mL | ||
| Enzyme Dilution Buffer, 5X | 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, 1 mg/mL BSA | 35 mL | ||
| EGTA Solution, 550 mM | 550 mM EGTA in 2 M NaOH | 0.15 mL | ||
| Europium Solution, | 30 mM Europium (III) in 0.6 mM HCl | 35 mL | ||
PhosphoSens-Kinetic Kinase Discovery Kits
PhosphoSens-Kinetic Kinase Assay Discovery Kits have sufficient volumes to perform 192 reactions in a low‐volume 384‐well plate (10μM sensor peptide in 25μL reaction volume) or 96 reactions in a 96-well plate (10μM sensor peptide in 50μL reaction volume).
*Discounted technology evaluation kits are available for first time users and for new projects. Please inquire.
| COMPONENT | DESCRIPTION | VOLUME |
| PhosphoSens Substrate, 1 mM | PhosphoSens Sensor Peptide Substrate, 1 mM | 60 µL |
| ATP Solution, 100 mM | 100 mM ATP in nuclease-free water | 60 µL |
| DTT Solution, 1 M | 1 000 mM DTT in nuclease-free water | 60 µL |
| Enzyme Reaction Buffer, 10X | 500 mM HEPES, pH 7.5, 0.1% Brij-35, 100 mM MgCl2 | 600 µL |
| Enzyme Dilution Buffer, 5X | 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, 1 mg/mL BSA | 1600 µL |
| EGTA Solution, 550 mM | 550 mM EGTA in 2000 mM NaOH | 60 µL |
PhosphoSens-Red Kinase Discovery Kit
PhosphoSens-Red Kinase Assay Kits have sufficient volumes to perform 96 reactions in a low‐volume 384‐well plate (10μM sensor peptide in 25μL reaction volume).
*Discounted technology evaluation kits are available for first time users and for new projects.
| COMPONENT | DESCRIPTION | VOLUME |
| PhosphoSens Substrate, 1 mM | PhosphoSens Sensor Peptide Substrate, 1 mM | 60 µL |
| ATP Solution, 100 mM | 100 mM ATP in nuclease-free water | 60 µL |
| DTT Solution, 1 M | 1 000 mM DTT in nuclease-free water | 60 µL |
| Enzyme Reaction Buffer, 10X | 500 mM HEPES, pH 7.5, 0.1% Brij-35, 100 mM MgCl2 | 600 µL |
| Enzyme Dilution Buffer, 5X | 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, 1 mg/mL BSA | 1600 µL |
| EGTA Solution, 550 mM | 550 mM EGTA in 2000 mM NaOH | 60 µL |
| Europium (III), 30 mM | 30mM Europium (III) in 0.6 mM HCl | 1200 µL |
PhosphoSens-Kinetic Phosphatase Discovery Kits
PhosphoSens-Kinetic Phosphatase Assay Discovery Kits have sufficient volumes to perform 192 reactions in a low‐volume 384‐well plate (10μM sensor peptide in 25μL reaction volume) or 96 reactions in a 96-well plate (10μM sensor peptide in 50μL reaction volume).
*Discounted technology evaluation kits are available for first time users and for new projects. Please inquire.
| COMPONENT | DESCRIPTION | VOLUME |
| PhosphoSens Substrate, 1 mM | PhosphoSens Sensor Peptide Substrate, 1 mM | 60 µL |
| DTT Solution, 1 M | 1 000 mM DTT in nuclease-free water | 60 µL |
| Enzyme Reaction Buffer, 10X | 500 mM HEPES, pH 7.5, 0.1% Brij-35, 100 mM MgCl2 | 600 µL |
| Enzyme Dilution Buffer, 5X | 20 mM HEPES, pH 7.5, 0.01% Brij-35, 5% Glycerol, 1 mg/mL BSA | 1600 µL |
| EGTA Solution, 550 mM | 550 mM EGTA in 2000 mM NaOH | 60 µL |
Three Enzyme Assay Families And A Robust Assay Generation Process
In addition to Continuous and Endpoint/Red Enzyme and Inhibitor Activity Assays, AssayQuant offers assays for three different families of enzymes: protein kinases, protein phosphatases and lipid kinases.
All AssayQuant protein kinase and phosphatase assays are based on the use of fluorogenic sensor-peptide-based substrates as described on our Technology page.
The lipid kinase (Diacylglycerol Kinases or DGKs), which use lipid (DAG) substrates, are currently only offered as part of AssayQuant’s Testing Services.
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