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Mouse phospho-SLP-76 (Ser376) cellular kit HTRF®

The mouse phospho-SLP-76 (Ser376) kit enables the cell-based quantitative detection of mouse phosphorylated SLP-76 at Ser376, for monitoring T-lymphocyte activation.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • No-wash No-wash
The mouse phospho-SLP-76 (Ser376) kit enables the cell-based quantitative detection of mouse phosphorylated SLP-76 at Ser376, for monitoring T-lymphocyte activation.
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Overview

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.

Benefits

  • SPECIFICITY
  • PRECISION

Mouse phospho-SLP-76 (Ser376) assay principle

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.

Phospho-SLP-76 (Ser376) assay principle

Mouse phospho-SLP-76 (Ser376) 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 the addition of mouse phospho-SLP-76 (Ser376) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-SLP-76 (Ser376) 2-plate assay protocol

Mouse phospho-SLP-76 (Ser376) 1-plate assay protocol

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.

Phospho-SLP-76 (Ser376) 1-plate assay protocol

TCR signaling stimulation with anti-CD3 antibodies in Jurkat cells

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.

SLP-76 stimulation in mouse EL-4 cells using anti-CD3 antibodies

Function and regulation of SLP-76

TCR engagement promotes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein tyrosine kinase (Lck). Zap-70 is recruited to the TCR/CD3 complex, where it becomes phosphorylated and activated. ZAP-70 phosphorylates SLP-76. Activated SLP-76 translocates to the plasma membrane and promotes a multi-protein complex, which participates in the activation, survival, and proliferation of T-Lymphocytes.
SLP-76 signaling pathway

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Plate Reader Requirement

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