Mouse phospho-SLP-76 (Ser376) cellular kit HTRF®
- High sensitivity
- Faster and more convenient than ELISA
This HTRF cell-based assay enables rapid, quantitative detection of mouse SLP-76 phosphorylated on Serine 376. Phospho-SLP-76 creates a scaffold on which key signaling complexes are built, and is a marker of T-lymphocyte activation.
The Mouse phospho-SLP-76 (Ser376) assay measures mouse SLP-76 when phosphorylated at Ser376. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Mouse phospho-SLP-76 (Ser376) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, and the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.
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 the addition of mouse phospho-SLP-76 (Ser376) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Detection of mouse phosphorylated SLP-76 (Ser376) 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.
Mouse EL-4 cells were plated in 6-well plates in complete culture medium, and incubated at 37°C, 5% CO2. The same day, cells were treated with 10 µg/mL of anti-CD3 antibody for 20 min, and then underwent a second treatment with 10 µg/mL of IgG with and without 11mM of H2O2 for 15min. After treatment, cells were then lysed with supplemented lysis buffer #1 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF mouse phospho-SLP76 (Ser376) or mouse Total-SLP76 detection reagents. The HTRF signal was recorded after ON incubation.
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
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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
63ADK109PEG-63ADK109PEH - Product Insert
Plate Reader Requirement
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