This HTRF kit monitors cellular BTK expression levels and can be used as a normalization assay with the phospho-BTK (Tyr223) kit.

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  • Ease-of-use Ease-of-use
  • Low sample consumption Low sample consumption

This HTRF kit monitors cellular BTK expression levels and can be used as a normalization assay with the phospho-BTK (Tyr223) kit.

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Overview

The Total BTK cellular assay monitors total BTK and is used as a normalization assay with our phospho-BTK kits with respect to steady state levels. 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 for X-linked agammaglobulinemia (XLA) disease.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-BTK assay principle

The Total-BTK assay quantifies the expression level of BTK in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-BTK assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of BTK in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
Total BTK assay principle

Total-BTK 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 Total BTK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total BTK 1-plate assay protocol

Total-BTK 1-plate assay protocol

Detection of total BTK 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.
Total BTK 2-plate assay protocol

HTRF assay compared to Western Blot using total BTK assay on human erythroleukemic K562 cell

Human erythroleukemic K562 cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2. After centrifugation, resuspension of pelleted cells in FBS-free medium (6 mL of medium containing 4 million cells/mL), and treatment with 100 µM pervanadate for 20 minutes at 37°C, cells were lysed with 3 mL of supplemented lysis buffer#1 for 30min at RT under gentle shaking. Soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRFtotal detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.
Side by side comparison of HTRF total BTK kit and WesternBlot

Accumulation of total and phosphorylated BTK (Tyr223) in human B-cell lymphoma line Raji

sing 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.
quantification of total BTK on human B-cell lymphoma line Raji

 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.
Validation of total BTK assay on Human Erythroleukemic K562 cells and Human lymphoma B-cell Raji cells

Inhibitory effect of P505-15, a Syk inhibitor 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.
Inhibitory effect of P505-15, a Syk inhibitor on the phosphorylation of BTK

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.
BTK signaling pathway
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - 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

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

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

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - 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

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

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

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 total Kit / 63ADK064PEG-63ADK064PEH

63ADK064PEG-63ADK064PEH - Product Insert

Cisbio Product Catalog 2019

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

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