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Phospho-SLP-76 (Ser376) & total SLP-76 Cellular Assay Kits Phospho- and total SLP-76 assays for monitoring T cell receptor signaling Cisbio's cell-based homogeneous HTRF® phospho-SLP-76 (Ser376) immunoassay enables the quantitative detection of SLP-76 phosphorylated on Serine 376. The HTRF® total SLP-76 assay kit is designed for the quantitative detection of total SLP-76, phosphorylated and unphosphorylated, to normalize the phosphorylation status of SLP-76 with respect to its steady-state level in cells. The buffers of both HTRF® phospho- and total SLP-76 assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample. SLP76 is known as an adaptor protein involved in signaling through the T cell antigen receptor complex and it is present in a number of hematopoietic cell lineages including T cells, platelets, neutrophils, mast cells, macrophages, and NK cells. T Cell Receptor (TCR) activation promotes a number of signaling cascades that ultimately determine cell fate through regulating cytokine production, cell survival, proliferation, and differentiation. Amenable to low-volume formats, HTRF® phospho-SLP-76 (Ser376) assay can be used in contexts ranging from basic research to high throughput drug screening.    Features Validated on Jurkat cell line lysates Low-volume samples required Truly homogeneous, simple protocol with no wash steps More convenient and faster than ELISA or Western Blot   Applications Functional screening for T cell receptor signaling Cellular pharmalogical quantification of total and phosphorylated SLP-76 (Ser376) Total SLP-76 for normalization      Assay Principle HTRF® - the homogeneous cell-based sandwich immunoassay Cisbio Bioassay’s phospho- and total SLP-76 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-SLP76 antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with SLP-76, and the proximity of donor and acceptor then leads 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 SLP-76 can be quantitatively detected using the HTRF phospho-SLP-76 (Ser376) and total SLP-76 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 first plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.          Product performance Validation of the HTRF phospho- and total SLP-76 assays on Jurkat cells Different densities (200K, 100K, 50K and 25K) of Jurkat cells were plated in suspension under 50µL in 96-well plates. The cells were stimulated with 25µL of increasing concentrations of anti CD3 antibody for 30 min at 37 °C - 5% CO2. After stimulation, cells were lysed with 25µL of lysis buffer 4X for 30 min at RT under gentle shaking. 16µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho SLP-76 (Ser376) or total SLP-76 detection reagents were added. The HTRF signal was recorded after a overnight incubation at room temperature.     

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