Total BTK cellular kit
Simple and robust detection kit for Phospho BTK & Total BTK
Phospho-BTK (Tyr223) cellular kit HTRF®
This HTRF kit enables the cell-based detection of Bruton tyrosine kinase (BTK) phosphorylation.
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Ease-of-use
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Low sample consumption
Overview
BTK is involved in the B cell receptor and the NF kappa B signaling pathways, and has an important role in primary immunodeficiency, in particular in X-linked agammaglobulinemia (XLA) disease. The phospho BTK assay enables detection of the Bruton tyrosine kinase phosphorylation on Tyrosine 223.
Benefits
- SPECIFICITY
- PRECISION
Phospho-BTK (Tyr223) assay principle
The Phospho-BTK (Tyr223) assay measures BTK when phosphorylated at Tyr223. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-BTK (Tyr223) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the proteins phosphorylation state under a no-wash assay format.
Phospho-BTK (Tyr223) 2-plate assay protocol
The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Phospho-BTK (Tyr223) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-BTK (Tyr223) 1-plate assay protocol
Detection of Phosphorylated BTK (Tyr223) with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
HTRF vs WB using phospho & total BTK assay on human K562 cells
Human erythroleukemic K562 cells were cultured for 2 days at 37°C, 5% CO2. After lysis and centrifugation, pelleted cells were resuspended in FBS-free medium treated with 100 µM pervanadate for 20 minutes. Cells were then lysed with and soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were indicated the 2 HTRF assays for either phospho or total BTK detections are 16-fold more sensitive than Western Blot.
Accumulation of total & phospho-BTK in B-cell lymphoma line Raji
Using the two-plate assay protocol, human B-cell lymphoma line Raji cells were plated at 200,000 cells/ well in a half 96 well plate in 20 µl of FBS-free culture medium. After treatment for 20 minutes at 37°C (in triplicate) with 10µL of pervanadate at diffent concentrations , cells were lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate with 4 µL of the pre-mix detection antibodies. The HTRF signal was recorded after an overnight incubation.
Inhibitory effect of Ibrutinib on the phosphorylation of BTK
Human Erythroleukemic K562 cells and Human lymphoma B-cell Raji cells were plated respectively at 100 kcells/well & 200 kcells/well under 20µl of FBS-free culture medium in a half-96 well plate. After treatment for 2 hours at 37°C with 5 µL of increasing concentrations of Ibrutinib, 5 µL of 50µM pervanadate were added for 20 min at 37°C. Cells were then lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate with 4 µL of the pre-mix detection antibodies. The HTRF signal was recorded after an overnight incubation.
Inhibitory effect of P505-15 on the phosphorylation of BTK
Human Erythroleukemic K562 cells and Human lymphoma B-cell Raji cells were plated respectively at 100 kcells/well & 200 kcells/well under 20µl of FBS-free culture medium in a half-96 well plate. After treatment for 2 hours at 37°C with 5 µL of increasing concentrations of P505-15, 5 µL of 50µM pervanadate were added for 20 min at 37°C. Cells were then lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate with 4 µL of the pre-mix detection antibodies. The HTRF signal was recorded after an overnight incubation.
BTK Simplified Pathway
BTK is a Cytoplasmic tyrosine kinase that's primarily expressed in B cells. It is involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells. Initiation of BCR signaling involves Lyn and Syk, part of the Src family. Lyn phosphorylates the intracellular domain of the BCR, leading to the recruitment and phosphorylation of Syk, and eventually, SLP65 phosphorylation. This leads to the recruitment and phosphorylation of BTK at Tyr551 and in turn, BTK autophosphorylates at Tyr223 for its full activation.
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Cisbio lysis buffer compatibility
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
PROTAC assays design at-a-glance
Streamlined set-up and access to multiple read-out options for research in oncology - Application Notes
PI3K/AKT/mTor translational control pathway - Posters
Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells
Analysis of a large panel of diverse biological samples and cellular models - Posters
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
Product Insert BTK P-Y223 Kit / 63ADK017PEG-63ADK017PEH
63ADK017PEG-63ADK017PEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
An innate and adaptive immunity recap
Insight into the diversity of cells & signaling pathways - Guides
Innovative solutions in Virology research
Kits and reagents for virology research - Flyers
Improve PROTAC Targeting Kinase Discovery
Application note describing how HTRF binding kits can help assessing PROTAC targeting kinase - Application Notes
Advance your research on Fibrosis
Kits and reagents for Fibrosis research - Flyers
Safety Data Sheet BTK P-Y223 Kit / 63ADK017PEG
63ADK017PEG - Safety Data Sheet
Safety Data Sheet BTK P-Y223 Kit / 63ADK017PEH
63ADK017PEH - Safety Data Sheet
Plate Reader Requirement
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