AssayQuant’s patented PhosphoSens® Technology forms the basis of its extensive portfolio of more than 400 unique enzyme and inhibitor assays. Each assay features novel sensor-peptide substrates available in the form of a kit or bulk sensor-peptide and as a family of related standalone reagents, each component is tested carefully to ensure that it is optimized for use in the applicable assay. Bulk reagents are available at discounted pricing, for high throughput or larger volume applications.
We offer two assay formats: Continuous (Fluorescence Intensity) and Red-Shift (Time Resolved Fluorescence). Both formats are compatible with 96, 384 and 1536-well plates and produce signals that are directly proportional to the amount of phosphorylated product present in the reaction mixture at the time of the measurement. In both cases, potency results have been shown to align with those obtained from cell-based assays, accelerating, and improving lead identification, characterization, profiling, and optimization studies, leading to the potential for developing drugs faster and with better efficacy.
Both assay formats are fully compatible with a wide range of ATP concentrations (including low to physiological [mM] levels) and use physiological Mg++, Mn++, Ca++ ion concentrations and peptide sequences derived from known substrates. With our selective PhosphoSens substrates, assays can be performed using crude cell or tissue lysates providing a more physiological context, including measuring activity of kinases in functional complexes that are formed in living cells.
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.
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.
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.