Continuous kinase enzyme & inhibitor activity assays: PhosphoSens

The PhosphoSens assay range from AssayQuant enables real-time observation of the entire reaction enabling easier K_i determination. Designed for simplicity and efficiency, the PhosphoSens® platform utilises a one-step, homogeneous fluorescence-based approach to rapidly and sensitively detect kinase activities for serine/threonine and tyrosine. By directly assessing kinase catalytic activity, PhosphoSens eliminates the need for antibody reagents, complex coupling procedures, or radioisotope labeling, streamlining the analysis and allowing for in-depth characterisation of inhibition dynamics. This technology has been featured in 135 publications so far.

Benefits of using PhosphoSens technology

• Capture the entire reaction in real-time
• Accurately determine kinetic parameters with a streamlined workflow
• Detect actual phosphorylation and dephosphorylation
• Compatible with any ATP concentration
• Obtain data using optimised physiologically relevant sensor peptides

PhosphoSens is available in multiple formats

• PhosphoSens-Kinetic assays use a simple add-and-read protocol for real-time, kinetic fluorescence intensity (FI) measurements. They offer high accuracy by measuring the initial reaction rate, with readings taken every 30 seconds to two minutes. The format corrects for compound autofluorescence by subtracting background signals from the total fluorescence. Compatible with most fluorescence microplate readers and lab automation, these assays require no stop solution, maximising throughput.
• PhosphoSens-Red assays detect fluorescence at longer wavelengths (>600 nm) using time-resolved fluorescence (TRF) to minimise signal contamination from auto-fluorescent compounds. With a recommended 100 µs delay and 300 µs acquisition time, these assays prevent interference from short-lived fluorescent materials. The same substrate used in continuous assays can often be applied, though for acidic substrates, alternative sensor-peptides are available for optimised performance, providing signal-to-background ratios of 2 or higher and Z' values of 0.7 or greater.
   

There are multiple advantages to kinome profiling with continuous assays

PhosphoSens provides both kinetic and endpoint readouts
Simply add Eu3+ to the kinetic assay to convert it to a Red-shifted endpoint assay

Applications
IC₅₀, EC₅₀ and K_i for catalytic inhibitor/activator potency  
Assays 
Kinome Profiling for compound selectivity 
MOI/MOA for inhibitor-substrate competition studies

Downloadable materials
PhosphoSens brochure
PhosphoSens manual
PhosphoSens publication list