Phospho-BTK (Tyr223) cellular kit
Simple and robust detection kit for Phospho BTK & Total BTK
Total SYK Cellular Kit HTRF®
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.
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No-wash
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High sensitivity
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All inclusive kit
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Low sample consumption
Overview
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.
Benefits
- SPECIFICITY
- PRECISION
- DATA NORMALIZATION
Total-SYK assay principle
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.
Total-SYK two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total-SYK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-SYK one-plate assay protocol
Detection of Total SYK 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.
SYK activation in anti-IgM-stimulated Ramos B cells
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.
SYK inhibition in macrophage-differentiated THP-1 cells treated with P505-15
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.
HTRF total SYK assay compared to Western Blot
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.
Simplified pathway of SYK signaling
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.
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In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
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Get the brochure about technology comparison. - Brochures
Product Insert SYK total Kit / 64SYKTPEG-64SYKTPEH
64SYKTPEG-64SYKTPEH - Product Insert
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On-demand webinar: Linking Neuroinflammation and Neurodegeneration
New insight into neuroinflammation research - Videos
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Safety Data Sheet (DEU) SYK total Kit / 64SYKTPEG
64SYKTPEG - Safety Data Sheet
Safety Data Sheet (ELL) SYK total Kit / 64SYKTPEG
64SYKTPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) SYK total Kit / 64SYKTPEG
64SYKTPEG - Safety Data Sheet
Safety Data Sheet (ITA) SYK total Kit / 64SYKTPEG
64SYKTPEG - Safety Data Sheet
Safety Data Sheet (SPA) SYK total Kit / 64SYKTPEG
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64SYKTPEG - Safety Data Sheet
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64SYKTPEG - Safety Data Sheet
Safety Data Sheet (DEU) SYK total Kit / 64SYKTPEH
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Safety Data Sheet (ELL) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
Safety Data Sheet (ITA) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
Safety Data Sheet (SPA) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) SYK total Kit / 64SYKTPEH
64SYKTPEH - Safety Data Sheet
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