The phospho-BTK (Tyr223) kit enables the cell-based detection of Bruton tyrosine kinase (BTK) phosphorylation.
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  • Ease-of-use
  • Low sample consumption
The phospho-BTK (Tyr223) kit enables the cell-based detection of Bruton tyrosine kinase (BTK) phosphorylation.
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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.

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 protein’s 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 96 well plate in 20 µl of FBS-free culture medium. After treatment for 20 minutes at 37°C (in triplicate) with increasing concentrations of pervanadate, the medium was removed and the 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 cultured and exposed to increasing concentrations of Ibrutinib and 5 µL of 50µM pervanadate. 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 cultured and exposed to 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

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

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

Do all cell-based kinase assays perform similarly? A side-by-side comparison of HTRF®, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? A side-by-side comparison of HTRF®, Western Blot, ELISA and AlphaScreen® SureFire® - 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

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

A guideline for 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

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

An indispensable complement to properly interpret 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

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

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Safety Data Sheet BTK P-Y223 Kit / 63ADK017PEG

63ADK017PEG - Safety Data Sheet

Safety Data Sheet BTK P-Y223 Kit / 63ADK017PEH

63ADK017PEH - Safety Data Sheet

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