Revvity, 64DAP12TPEH, HTRF Human Total DAP12 Detection Kit, 10,000 assay points

Overview
The Total DAP12 cellular assay monitors total DAP12 protein levels and can be used as a normalization assay in conjunction with our Phospho-DAP12 (Tyr91) kit. This kit is designed to be compatible with the buffers from the Phospho-DAP12 kit, allowing the same lysate to be used for analyses of both the phosphorylated and the total protein populations. 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.
The DAP12-TREM2 signaling axis is particularly important in the context of neuroinflammation and neurodegenerative diseases. It regulates microglial activation and function, influencing the brain's immune response and homeostasis. Genetic variants of TREM2 have been identified as risk factors for Alzheimer's Disease, highlighting the significance of this pathway in maintaining brain health. Monitoring total DAP12 levels alongside its phosphorylated form provides valuable insights into the overall expression and activation state of this crucial signaling protein.
How it works
Total DAP12 assay principle
The Total DAP12 assay quantifies the expression level of DAP12 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total DAP12 assay uses two labeled antibodies, one coupled to a donor fluorophore and the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of DAP12 this 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 total protein present in the sample and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Total DAP12 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 HTRF Total DAP12 detection reagents. This protocol allows for the cells' viability and confluence to be monitored.

Total DAP12 one-plate assay protocol
Detection of Total DAP12 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-Total
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