Total BTK cellular kit HTRF®

Associated products

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

Ease-of-use
Low sample consumption

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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 proteins expression under a no-wash assay format.

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

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.

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

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 different 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.

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.

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

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

PROTAC assays design at-a-glance

Streamlined set-up and access to multiple read-out options for research in oncology - Application Notes

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

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

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

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 total Kit / 63ADK064PEG-63ADK064PEH

63ADK064PEG-63ADK064PEH - 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

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

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

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

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

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Safety Data Sheet (DEU) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (ELL) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (FRA-FR) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (ITA) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (SPA) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (ENG-GB) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (ENG-US) BTK total Kit / 63ADK064PEG

63ADK064PEG - Safety Data Sheet

Safety Data Sheet (DEU) BTK total Kit / 63ADK064PEH