Mouse Total SLP-76 cellular kit HTRF®

The Mouse Total SLP-76 kit detects cellular SLP-76, and can be used as a normalization assay with our Mouse phospho-SLP-76 kit to assist in the optimal monitoring of T-lymphocyte activation.

Ready-to-use
High sensitivity
Faster and more convenient than ELISA
No-wash

Download the protocols

The Mouse Total SLP-76 kit detects cellular SLP-76, and can be used as a normalization assay with our Mouse phospho-SLP-76 kit to assist in the optimal monitoring of T-lymphocyte activation.

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Overview

The Mouse Total SLP-76 cellular assay kit is designed to monitor the expression level of SLP-76, whether phosphorylated or unphosphorylated. It is compatible with our Mouse phospho-SLP-76 (Ser376) kit, and enables the analysis of phosphorylated and total proteins from a single sample for better readouts of T-lymphocyte activation.

Benefits

SPECIFICITY
PRECISION
DATA NORMALIZATION

Total-SLP-76 assay principle

The Total-SLP-76 assay quantifies the expression level of SLP-76 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-SLP-76 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SLP-76 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-SLP-76 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 total SLP-76 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Total-SLP-76 1-plate assay protocol

Detection of mouse total SLP-76 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.

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, the 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 this 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.

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Plate Reader Requirement

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