Revvity, 64DAP12Y1PEH, HTRF Human Phospho-DAP12 (Tyr91) Detection Kit, 10,000 assay points

Overview
This HTRF cell-based assay provides a convenient and accurate method for detecting phosphorylated DAP12 at Tyr91. DAP12 (DNAX-Activation Protein 12), also known as TYROBP (TYRO protein tyrosine kinase-binding protein), is a transmembrane signaling adaptor protein that plays a crucial role in innate immune responses and neuroinflammation. DAP12 is widely expressed in various immune cells, including natural killer cells, monocytes, macrophages, and microglia, the resident immune cells of the central nervous system. In the context of neuroinflammation, DAP12 is particularly important due to its association with the TREM2 (Triggering Receptor Expressed on Myeloid cells 2) receptor. The DAP12-TREM2 signaling axis is a key regulator of microglial function, influencing processes such as phagocytosis, inflammatory responses, and lipid metabolism. By enabling the detection of phosphorylated DAP12 at Tyr91, this assay provides researchers with a valuable tool for investigating microglial activation, neuroinflammatory processes, and the potential effects of therapeutic interventions targeting the DAP12-TREM2 pathway.
How it works
Phospho-DAP12 (Tyr91) assay principle
The Phospho-DAP12 (Tyr91) assay measures DAP12 when phosphorylated at Tyr91. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other 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 independently 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-DAP12 (Tyr91) two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-DAP12 (Tyr91) HTRF detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Phospho-DAP12 (Tyr91) one-plate assay protocol
Detection of Phosphorylated DAP12 (Tyr91) 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 allows miniaturization while maintaining robust HTRF quality. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 3
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target-DAP12
Target Class-Phosphoproteins
Target Species-Human
Technology-TR-FRET
Therapeutic Area-Central Nervous System
Unit Size-10,000 assay points