PhosphoSens®-Lysate Kinase Assays
Measure real-time kinase activity directly from cell extracts and tissue homogenates
Real-Time Kinase Activity Analysis in Complex Cellular Contexts
The ~500 protein kinases in the human genome are vital for cellular function, and their dysregulation drives over 400 diseases, including cancer and inflammatory diseases. While 82 kinase inhibitors are approved, and AI/ML promises acceleration of new inhibitors, many drugs optimized against recombinant proteins show poor efficacy in patients. This is because these drug candidates are typically screened and validated in a cell-free context, losing important signaling complexes found in cells.
PhosphoSens-Lysate Assays transform kinase activity measurement by enabling continuous, real-time kinetic analysis directly in cell and tissue lysates. This innovative approach provides key insights into your target’s native environment throughout the drug discovery pipeline and uniquely enables:
- A deeper understanding of complex kinase-dependent signaling networks
- Ultra high-throughput kinase inhibitor screening in a physiologically relevant context
- Enhanced assay sensitivity for drug discovery and translational research
- Development of improved therapeutics with superior pharmacological profiles
PhosphoSens-Lysate: The Competitive Edge in Kinase Research
Improve Results by Assessing Activity of Your Target Kinase in Cell and Tissue Lysates Throughout the Drug Discovery Pipeline
Simplify and Accelerate your Research and Drug Development
Applicable from early discovery through preclinical validation, PhosphoSens-Lysate assays provide deep enzymological data and functional protein readout that uniquely enable a deeper understanding of kinase biology and development of improved drugs with superior pharmacological profiles.
Basic Research and Target Validation
Drug Discovery & Inhibitor Screening
Lead Identification and Optimization
Investigational New Drug (IND) Enabling Studies
Benefits Over Recombinant Kinase Assays
By using unfractionated cell lysates or tissue homogenates, PhosphoSens lysate assays provide a closer approximation of the native kinase environment, allowing you to measure catalytic activity in a direct, continuous, quantitative, and easy-to-use format. Our direct lysate activity assays combine the efficiency of biochemical testing/screening with the native complexity of the cellular milieu including the following factors, all of which can significantly impact kinase biology and inhibitor performance in vivo:
Cellular components
Native proteins (full-length, no tags), lipids, nucleic acids, small molecules, cofactors and binding partners that form functional complexes.
Post-translational modifications (PTMs)
An abundance of PTMs that can influence kinase activity, localization, and interactions, providing physiologically relevant insights.
Cellular signaling networks
Native complexes, regulatory feedback loops, and signaling crosstalk between pathways are critical to understanding normal and disease states and effective drug development.
Competing enzymes
Potential interfering kinases, phosphatases, and proteins that are important to screen against for potency and selectivity.
Available Now For ERK1/2!
Designed to measure Extracellular signal-Regulated Kinase1/2 (ERK1/2, Common names) or Mitogen-activated Protein Kinase 3/1 (MAPK3/1, HGNC names) activity directly in cell and tissue lysates, this groundbreaking assay provides unparalleled precision and efficiency for studying these kinases in complex samples .
We are developing Selective Substrates for the following targets:
MEK1/2, PIM1/3, JNK1-3, P38a/b/g/d, GSK3a/b, AKT1-3, CDK1-3/5, and CDK4/6.
Interested in learning more about ERK1/2 activity assays?
Access our ERK1/2 PhosphoSens-Lysate Assay Validation Report for a detailed overview of the experiments conducted to ensure optimal assay performance, reliability, and reproducibility.
How the PhosphoSens-Lysate Assay Works
PhosphoSens-Lysate Assay leverages AssayQuant’s Proprietary Detection Technology to provide real-time, continuous measurements of kinase activity by directly detecting substrate phosphorylation throughout the kinase reaction.
Target Selective Sensor Peptide Substrates are developed with our readout molecule, Sulfonamido-oxine (Sox), covalently attached via a cysteine residue (native or introduced) in close proximity to the Serine, Threonine, or Tyrosine phosphorylation site (± 2-5 residues).
The Sox fluorophore reports peptide phosphorylation by the target kinase via chelation-enhanced fluorescence (ChEF). The fluorescence-sensing mechanism is based on the chelate effect introduced by phosphorylation of the Ser/Thr/Tyr residue proximal to CSox, which enhances magnesium (Mg²⁺) binding affinity compared to the unphosphorylated species, and results in increased fluorescence emission intensity (2-10-fold).
In this way, CSox-modified peptide substrates provide a fluorescent intensity signal that is directly proportional to the phosphorylation level of the substrate, enabling continuous/real-time kinetic measurements of kinase activity in medium to high throughput (96−1536 well plate).
Prepare your Sample
Grow cells (+/- stimulus), aspirate medium and wash, and add ice-cold extraction buffer with protease and phosphatase inhibitors to lyse the cells.
Start Kinase Reaction
Sample is added to reaction mix containing Target-Selective Sensor Peptide Substrate, catalyzing phosphotransfer from ATP to the substrate in real time.
Measure Kinase Activity
Add plate to reader and monitor kinase activity throughout the reaction. Signal is directly proportional to the phosphorylation level of the substrate.
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PhosphoSens-Lysate Discovery Kits
Designed for technology evaluation, our Lysate Discovery Kits come complete with a target selective substrate and sufficient kinase reaction reagents for 200 kinetic assays.
We also provide a small sample of recombinant protein as a control and our optimized PhosphoPreserve Cell Extraction Buffer so you can evaluate the technology using your own cells!
PhosphoSens Selective Substrates
Designed for target specific activity analysis in lysates, these selective substrates can be purchased as lyophilized 1mg aliquots. Each mg is sufficient for ~2,500 assays in a low‐volume 384‐well plate (10μM sensor peptide in 25μL reaction volume).
Selective Substrate PhosphoControls
Fully phosphorylated selective substrates can be purchased as lyophilized 1mg aliquots. Each mg is sufficient for ~2,500 assays in a low‐volume 384‐well plate (10μM sensor peptide in 25μL reaction volume).
PhosphoPreserve Cell Extraction Buffer
This formulation has been optimized to preserve the activity of kinases in crude cell or tissue lysates. Please note that it is important to add the protease and phosphatase inhibitors to the extraction buffer just before use.
How to Order
Kinase assays via credit card
Order PhosphoSens Substrates, Substrate Systems, and Discovery Kits online via credit card
Phosphatase assays via credit card
Order PhosphoSens Substrates, Substrate Systems, and Discovery Kits online via credit card
Kinase and Phosphatase assays via purchase order
Inquire about purchasing any of our PhosphoSens Products or KinSight Services
Didn’t find what you were looking for?
We also offer custom assay development and kinase and phosphatase profiling services. Tell us about your specific project, and our experts can help you select the right PhosphoSens tool for your kinetic assay needs.
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