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

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.