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About BTK
Bruton tyrosine kinase (BTK) is a non-receptor tyrosine kinase and founding member of the Tec family, which also includes ITK, TEC, BMX, and TXK. BTK is activated downstream of the B-cell receptor (BCR) through a two-step mechanism: first, LYN-mediated phosphorylation of the PH domain recruits BTK to the plasma membrane, where PI3K-generated PIP3 promotes autophosphorylation at Y551 in the activation loop by SYK, followed by autophosphorylation at Y223 in the SH3 domain. Key substrates include PLCγ2, which drives calcium mobilization, PKCβ, and NF-κB pathway effectors. BTK is indispensable for BCR signaling, B-cell development, differentiation, and survival. Dysregulation drives B-cell malignancies including CLL, MCL, DLBCL, and Waldenström macroglobulinemia, as well as autoimmune diseases such as rheumatoid arthritis and lupus. Loss-of-function mutations cause X-linked agammaglobulinemia (XLA). BTK's central role in oncogenic BCR signaling, its druggable ATP-binding pocket featuring a unique cysteine (C481), and its restricted hematopoietic expression make it a premier oncology and immunology drug target.
Why PhosphoSens for BTK?
BTK assay development is complicated by its dual-substrate kinetics (PLCγ2-derived peptides vs. full-length substrates), the prevalence of covalent irreversible and reversible-covalent inhibitors targeting C481, and artifacts introduced by endpoint assays when measuring covalent inactivation. ADP-Glo and HTRF methods cannot distinguish time-dependent inhibition from equilibrium binding and require quenching steps that obscure kinact/KI parameters critical for covalent drug characterization. Radiometric assays offer sensitivity but lack real-time resolution. PhosphoSens continuous fluorescent kinase assays use chelation-enhanced fluorescent (CSox) peptide substrates to generate full progress curves at physiological ATP concentrations, enabling direct extraction of kinact/KI for covalent BTK inhibitors like ibrutinib, acalabrutinib, and zanubrutinib. Real-time monitoring eliminates endpoint artifacts, supports compound selectivity profiling, and dramatically accelerates SAR campaigns.
Key Pathways:
B-cell receptor (BCR) signaling
NF-κB signaling
PI3K-AKT signaling
PLCγ2-calcium-PKC signaling
Approved Inhibitors:
Ibrutinib (Imbruvica, CLL/MCL/WM/MZL/CLL with 17p deletion/cGVHD
Acalabrutinib (Calquence, CLL/SLL/MCL
Zanubrutinib (Brukinsa, CLL/SLL/MCL/WM/MZL/FL
Pirtobrutinib (Jaypirca, MCL/CLL/SLL)
Talk to a Scientist
Have questions about BTK assay design, selectivity panels, or covalent inhibitor characterization?
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How pricing works:
Select a kit or substrate below, then request a quote. Bundle discounts apply automatically.
Select a kit or substrate below, then request a quote. Bundle discounts apply automatically.
Real-Time BTK Activity Assays
Continuous, real-time fluorescent assays optimized for quantitative BTK activity measurements, IC50 determination, and mechanistic studies.
Assay Format: Continuous kinetic fluorescence (real-time)
PhosphoSens-Kinetic assays directly quantify enzyme activity by continuously monitoring substrate phosphorylation or dephosphorylation in real time, generating a full progress curve in every well.
Learn more about PhosphoSens-Kinetic →Need pricing or availability? Select a kit or substrate to request a quote below.
Kits
PhosphoSens-Kinetic Kinase Activity Kit - AQT0101
Ready-to-use assay kits containing substrate and all essential reagents.
Kit Contents
- Optimized PhosphoSens® substrate (AQT0101)
- ATP, DTT, EGTA, Reaction Buffer & Dilution Buffer
Automatically save 10% when bundling 10ug recombinant enzyme with your 1,000 assay kit. View enzymes
Price: $425
Substrate
PhosphoSens Kinase Substrate AQT0101
Bulk PhosphoSens® substrate for assay development and high-throughput workflows.
Specifications
Automatically save 10% when bundling Reagent Packs with your substrate. View Reagent Packs
Price: $2795
Ready to request pricing for BTK?
Select a kit, substrate, or enzyme above. Our team will confirm pricing, availability, and any applicable bundle discounts.
Request a QuoteTRF BTK Screening Assays
Robust endpoint TRF assays optimized for high-throughput screening and quantitative BTK activity measurement in plate-based workflows.
Assay Format: Time-resolved fluorescence (TRF, endpoint)
PhosphoSens-Red assays use europium-based time-resolved fluorescence detection to enable robust, plate-based quantification of enzyme activity, making them well suited for high-throughput screening workflows.
Learn more about PhosphoSens-Red →Need pricing or availability? Select a kit or substrate to request a quote below.
Kits
PhosphoSens-Red Kinase Activity Kit - AQT0663
Ready-to-use TRF assay kits containing substrate and essential reagents.
Kit Contents
- Optimized PhosphoSens® substrate (AQT0663)
- ATP, DTT, EGTA, Reaction Buffer & Dilution Buffer
- Europium Detection Solution
Automatically save 10% when bundling 10ug recombinant enzyme with your 1,000 assay kit. View enzymes
Price: $455
Substrate
PhosphoSens Kinase Substrate AQT0663
Bulk substrate for PhosphoSens-Red TRF assays and high-throughput workflows.
Specifications
Automatically save 10% when bundling Reagent Packs with your substrate. View Reagent Packs
Price: $2795
Ready to request pricing for BTK?
Select a kit, substrate, or enzyme above. Our team will confirm pricing, availability, and any applicable bundle discounts.
Request a QuoteActive Recombinant Enzymes for BTK
Wild-type and mutant forms of BTK for assay development, kinase profiling, and mechanistic studies. Enzymes are supplied active and optimized for PhosphoSens® substrates.
BTK, Active Full Length
Rat • Baculovirus-Insect Cells
Technical Specifications
- Catalog: AQT-B10-10CG
- Mutation: Full Length
- Species: Rat
- Expression System: Baculovirus-Insect Cells
- Storage Buffer: Supplied as sterile 50 mM Tris-HCl, pH 7.5, 50-300mM NaCl, 10mM glutathione, 0.1mM EDTA, 0.25-1.0mM DTT, 0-0.1mM PMSF, 10-25% glycerol
- Molecular Weight: ~105 kDa
- Purity/Activity: For specific information on a given lot, see related technical data sheet.
- Storage Conditions: Store product at -70°C. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.
Available Sizes:
Price: $$348.00
Request a QuoteFrequently Asked Questions: BTK Assays
How does PhosphoSens measure covalent BTK inhibitor kinetics such as kinact/KI?
PhosphoSens continuous assays monitor substrate phosphorylation in real time, generating full progress curves that directly capture the time-dependent loss of BTK activity caused by covalent inactivators. By fitting progress curves at multiple inhibitor concentrations, kinact and KI can be extracted in a single experiment without the endpoint artifacts that confound ADP-Glo or HTRF approaches. This is essential for characterizing irreversible inhibitors like ibrutinib and acalabrutinib and for distinguishing covalent from non-covalent binding modes.
What peptide substrates are available for BTK PhosphoSens assays?
AssayQuant offers optimized CSox-based peptide substrates derived from BTK's physiological substrate PLCu03b32, engineered to balance Km, fluorescence dynamic range, and aqueous solubility. These substrates are validated at physiological ATP concentrations (matching cellular Km,ATP ~1 mM) to provide pharmacologically relevant IC50 values that correlate with cellular potency. Custom substrate development is also available for specific research needs.
Can PhosphoSens BTK assays be used for selectivity profiling against other Tec family kinases?
Yes, AssayQuant provides continuous kinase assays for multiple Tec family members including ITK and TEC, enabling direct head-to-head selectivity profiling under identical assay conditions. Because all assays use the same PhosphoSens platform with physiological ATP, IC50 values are directly comparable across kinases without normalization artifacts introduced by different detection chemistries. This is particularly valuable for distinguishing BTK-selective inhibitors from pan-Tec inhibitors in the context of autoimmune drug discovery.
Data & Resources
Explore data and documents to support your kinase and phosphatase experiments. Download sample data, protocols and other resources to see how our assays perform and to help you get started in your own lab. All validation data generated using PhosphoSens® assays under recommended conditions.
Sample Report: AQT0734 - EGFR Assay Validation Report
Each validation report provides experimental conditions and data showing:
- Enzyme titration and linearity
- Sensor peptide substrate titration and Km determination
- ATP titration and Km determination
- DMSO titration and assay tolerance
- Inhibitor titration and IC50 determination
Protocol
Inhibitor IC50 Protocol
See how the PhosphoSens-Kinetic Assay can be used to find the IC50 of a kinase inhibitor.
Download Protocol →Webinar
Webinar: Continuous Assay Validation
Discover how continuous assay formats power deep understanding of kinase function. See how PhosphoSens® assays guide inhibitor profiling, selectivity assessment, and mechanistic characterization.
Watch the Webinar →Service
KinSight™ BTK Profiling
Need broader selectivity data? KinSight profiling runs your compounds across our full kinase panel under identical PhosphoSens conditions.
Learn about KinSight →Stay Informed
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