Phospho-BTK (Tyr223) cellular kit
Simple and robust detection kit for Phospho BTK & Total BTK
The Total SYK kit is designed to monitor the expression level of cellular SYK (Spleen Tyrosine Kinase), and can be used as a normalization assay for the Phospho-SYK (Tyr525/526) kit.
The Total SYK cellular assay monitors total SYK, and can be used as a normalization assay with our phospho-SYK (Tyr525/526) kit. This kit is compatible with the buffers from the phospho-SYK kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
SYK (Spleen Tyrosine Kinase) is a cytoplasmic tyrosine/serine kinase which plays a crucial role in signal transduction, especially in immune cells such as B cells, macrophages, microglia (brain-resident macrophages), neutrophils and mast cells. Upon cell stimulation, SYK interacts with the cytoplasmic domains of activated immune receptors and binds to the specific phosphorylated receptor motifs (called "p-ITAMs"), leading to its activation by auto-phosphorylation of its catalytic domain at Tyr525/526.
SYK phosphorylation is a readout of key inflammatory pathways such as BCR, FcR and TREM2 signaling pathways that are altered in various pathologies such as auto-immune diseases, neurodegeneration, cancers and allergic disorders.
The Total SYK assay quantifies the expression level of SYK in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-SYK assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In the presence of SYK 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.
The human B cell lymphoma Ramos cell line was seeded in a half 96-well culture-treated plate at 100,000 cells/well in 20 µL FBS-free culture medium. After an overnight incubation at 37 °C, 5% CO2, cells were stimulated with 10 µL of increasing concentrations of an anti-human IgM antibody for 10 minutes, and then lyzed with 10 µL of supplemented lysis buffer #3 (4X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-SYK (Tyr525/526) or Total-SYK detection reagents were added. The HTRF signal was recorded after an overnight incubation.
The anti-human IgM antibody induced a dose-dependent increase in SYK phosphorylation at Tyr525/526 without changing the expression level of the protein, demonstrating the activation of the BCR complex at the cell surface.
The human monocytic THP-1 cell line was seeded in a 96-well culture-treated plate at 200,000 cells/well and differentiated into macrophages by incubation with 100 ng/mL PMA for 24h at 37 °C, 5% CO2. After differentiation, cells were first treated with increasing doses of the SYK inhibitor P505-15 for 1h, and then with 250 µM pervanadate for 10 minutes. After treatment, cells were lyzed with 50 µL of supplemented lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking, and 16 µL of cell lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho-SYK (Tyr525/526) or Total-SYK detection reagents. The HTRF signal was recorded after an overnight incubation.
As expected, the SYK inhibitor P505-15 induced a dose-dependent decrease in SYK phosphorylation at Tyr525/526, while the expression level of the kinase was not modulated by the treatment.
The human B cell lymphoma Ramos cell line was cultured in a T175 flask in complete culture medium for 48h at 37 °C, 5% CO2. After centrifugation, pelleted cells were stimulated with 20 µg/mL of an anti-human IgM antibody for 10 minutes, and after an additional centrifugation step, cells were lyzed with 10 mL of supplemented lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking.
Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF total-SYK detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.
Using the HTRF total-SYK assay, 500 cells/well were enough to detect a significant signal, while 2,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF total-SYK assay was 4 times more sensitive than the Western Blot technique.
SYK (Spleen Tyrosine Kinase) is a cytoplasmic tyrosine/serine kinase which plays a crucial role in signal transduction, especially in immune cells such as B cells and microglia (brain-resident macrophages).
Upon stimulation of B cell or TREM2/DAP12 receptor complexes, SYK interacts with the cytoplasmic domains of CD79 subunits or DAP12 respectively by binding to their specific phosphorylated receptor motifs, called 'p-ITAMs'. This leads to SYK activation by auto-phosphorylation of its catalytic domain at Tyr525/526. SYK in turn phosphorylates downstream substrates such as BTK, triggering the activation of multiple signaling pathways and cellular responses, including cell activation, proliferation, and survival.
Physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Get the brochure about technology comparison. - Brochures
See published experiments and data demonstrating how HTRF rises to the challenge of studying microglia in neuroinflammation research. - Application Notes
New insight into neuroinflammation research - Videos
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