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HTRF Human Total BTK Detection Kit, 10,000 Assay Points
This HTRF kit monitors cellular BTK expression levels and can be used as a normalization assay with the phospho-BTK (Tyr223) kit.
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption & disposal requirements under European REACH regulations (EC 1907/2006).
Product Variants
Product information
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.
Specifications
| Assay Points |
10000
|
|---|---|
| Assay Target Type |
Kit
|
| Assay Technology |
HTRF
|
| Brand |
HTRF
|
| Quantity |
1
|
| Therapeutic Area |
Oncology & Inflammation
|
| Unit Size |
10,000 Assay Points
|
Video gallery
How it works
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.
Assay validation
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.
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.
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, 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.
Simplified pathway
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.
Resources
Application Note
Enhance PROTAC Drug Discovery with AlphaLISA and HTRF Kits
A Bruton Tyrosine Kinase (BTK) case study
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Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
Flyer
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