Our rigorous development, validation and manufacturing practices create assays that are easier, faster and more informative.
AssayQuant is a life science company focused on developing innovative kinase and phosphatase enzyme assay formats and providing enabling screening services using its proprietary PhosphoSens® Platform. Through rigorous development, validation and manufacturing practices we create products that deliver exceptional value while providing unmatched results. This homogeneous and continuous (kinetic) format can be used with physiological (mM) ATP and with both purified enzymes and crude lysates.
Our PhosphoSens Platform harnesses the power of Chelation-Enhanced Fluorescence (ChEF) with improved sulfonamido-oxine (Sox) chromophore technology developed by the Imperiali laboratory and exclusively licensed from the Massachusetts Institute of Technology (MIT). These improvements include the cysteine-Sox method that allows for flanking sequence recognition determinants on either side of the Sox moiety/phosphorylation site to create superior peptide sensors. The result is a simple yet powerful method to measure the activity of kinases or phosphatases using a homogeneous and direct format. The level of fluorescence in this format is directly proportional to the amount of phosphorylated product, where optimized sensor sequences provide highly sensitive detection of the catalytic activity of the target enzymes.
Our PhosphoSens Platform offers unmatched features and benefits, including the ability to use the same optimized sensor in two formats - a continuous (kinetic) assay using fluorescence intensity (Excitation Max 360 nm & Emission Max 485 nm), or an endpoint-Red assay using Europium (Eu3+) with time-resolved fluorescence (Excitation Max 360 nm & Emission Max 620 nm).
Our continuous (kinetic) format is ideal for elucidating drug mechanism of action, potency and profiling. This continuous (kinetic) format is based on the much higher affinity of Mg2+ for the CSox-phosphopeptide compared to the corresponding non-phosphopeptide. Upon phosphorylation at the targeted serine, threonine or tyrosine residue and at physiological Mg2+ concentrations (10-20 mM), there is an immediate increase (2-10-fold) in fluorescence of the sensor, where continued phosphorylation in the reaction over time results in a linear progress curve (determined after subtracting background fluorescence at each time point).
The same principle of our kinetic format works for protein phosphatases, but monitoring decrease in fluorescence. Using the PhosphoSens Platform have developed a panel of CSox-based phosphopeptides derived from physiological substrates, where the activity of these enzymes can be monitored by the loss in fluorescence signal due to dephosphorylation of the substrate sensor over time.
At any point in time, Europium (Eu3+) can be added directly to the reaction to create a time-resolved fluorescence readout. In this format, excitation of the Sox fluorophore serves to sensitize the Europium luminescence, resulting in an emission signal maximum at 620 nm, which combined with a 100 μs delay, eliminates interference due to compound autofluorescence. PhosphoSens-Red is ideal for high-throughput screening (HTS) and structure-activity-relationships (SAR) applications, where there are large numbers of tests read in batch mode and where compounds may exhibit autofluorescence that interferes with the detection step of the assay.
Our optimized assay development process harnesses the many advantages of the PhosphoSens platform, allowing us to rapidly develop robust Sox-based peptide sensor assays even for kinases that have typically used protein substrates. We can apply our approach to develop custom assays for your organization for both highly generic substrates for use with a range of purified kinases or highly selective substrates for use with crude lysates.
Leveraging our rigorous sensor optimization process to develop highly selective sensors for your target enzyme of interest, provides the opportunity to measure kinase or phosphatase activity in unfractionated lysates made from cells or tissues. This feature allows drug development and screening to be conducted in a more physiological context where the target is found in the relevant signaling complexes (e.g., Li et al., 2009; Stains et al. 2012; Peterson et al., 2014; Broughton et al., 2019). Please see the literature section under resources for additional information.
Continuous detection of the activity of DGKs in lipid-micelles. DGKs are an important class of lipid kinases that catalyze the phosphorylation of numerous diacylglycerol (DAG) substrates to create phosphatidic acid (PA). Instead of using a Sox-based peptide substrate, AssayQuant has adapted the PhosphoSens format to incorporate DAG as the substrate and where PA then is monitored using the same direct, continuous (kinetic) and quantitative assay format. Currently, we have assays for 8 of the 10 DGK isoforms. These assays are only offered as part of our Custom Testing Services, which includes potency, profiling and mechanism of action, versus ATP and DAG, assessments to enable the generation of DGK inhibitors.
Our fit-for-purpose sensors and multiple assay formats provide seamless integration, improving efficiency and results with your drug development efforts. With three formats, PhosphoSens®-Red, Kinetic and Lysate, our Platform enables your drug development workflow to address the challenges and opportunities for next-generation kinase and phosphatase inhibitors or activators.
The Sox technology is covered by several patents and has been exclusively licensed by MIT to AssayQuant Technologies, Inc.
AssayQuant is a life science company founded by scientists who are committed to innovation. Through our advancements in assay design, we are transforming the way science gets done to deliver breakthrough discoveries needed to improve human health.