Phospho-BTK (Tyr223) & Total BTK Cellular Assay Kits

Phospho- and total BTK assays for monitoring BTK pathway

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. Cisbio's cell-based homogeneous HTRF® phospho-BTK (Tyr223) immunoassay enables the detection of the Bruton tyrosine kinase phosphorylation on Tyrosine 223. The HTRF® total BTK assay kit is designed for the quantitative detection of total BTK, phosphorylated and unphosphorylated, to normalize the phosphorylation status of BTK with respect to its steady-state level in cells.

   Check our lysis buffer compatibility

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio Bioassay’s phospho- and total BTK assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-BTK antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with BTK, and the proximity of donor and acceptor will then lead to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-BTK can be quantitatively detected using the HTRF phospho-BTK (Tyr223) and total BTK cellular assay kit reagents, and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

What to expect at the bench

This video covers the principles of phospho-HTRF assays and gives guidelines for performing them, using our phospho-ERK assay as an example.

Simplified pathway

BTK = Bruton’s Tyrosine Kinase (also known as “agammaglobulinaemia tyrosine kinase”) is a Cytoplasmic tyrosine kinase, member of the TEC kinase family, primarily expressed in B cells, but also in mast cells, platelets, and myeloid cells (not expressed in T cells, natural killer cells and plasma cells).

BTK is involved in multiple signal-transduction pathways regulating survival, activation, proliferation, and differentiation of B-lineage lymphoid cells.
Initiation of BCR (B cell receptor) signaling involves Lyn and Syk, part of the the Src family. Lyn phosphorylates the intracellular domain of the BCR, leading to the recruitment and phosphorylation of Syk. Activated Syk then phosphorylates the adaptor protein SLP65 (also known as BLNK, for B-cell linker protein), which leads to the recruitment of BTK and its phosphorylation by Syk at Tyr551. BTK in turn autophosphorylates at Tyr223 for its full activation, and then phosphorylates PLC-γ2.
Activation of PLC-γ2 leads to the generation of the downstream second messengers DA (diacylglycerol) and IP3, inducing the release of intracellular Ca2+ and the activation of PKC-β. These in turn activate the transcription factors NFAT (nuclear factor of activated T cells) and NFκB, as well as the MAP kinases ERK1/2, JNK and p38.
Lyn also activates the BCR coreceptor CD19, leading to the activation of the PI3K/AKT pathway responsible for B-cell survival and proliferation.

BTK is overexpressed and constitutively active in non-Hodgkin’s lymphoma, cell lymphoma and chronic lymphocytic leukemia, and is a therapeutic target for the screening of small molecule inhibitors to treat cancer.
Due to a systemic upregulation of B-cells, BTK is involved in autoimmune diseases, e.g. rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE).
Due to loss-of-function mutations of BTK, it is involved in B-cell immunodeficiency, e.g. X-linked agammaglobulinemia (XLA).  

Product Performance

1. HTRF assay compared to Western Blot using phospho and total BTK assay on human erythroleukemic K562 cells

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 HTRF phospho and total detection reagents. Equal amounts of lysates were used for a side by side comparison between Western Blot and HTRF.

The 2 HTRF assays for either phospho or total BTK detections are 16-fold more sensitive than Western Blot.

2. 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 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.

3. 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.

4. 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.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
BTK phospho-Y223 kit - 500 tests63ADK017PEG
BTK phospho-Y223 kit - 10,000 tests63ADK017PEH
BTK total kit - 500 tests63ADK064PEG
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BTK total kit - 10,000 tests63ADK064PEH
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Companion products

DescriptionCat. noProduct insertMSDS
BTK phospho-Y223 kit - control lysate63ADK017TDA
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BTK total kit kit - control lysate63ADK064TDA
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Literature