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

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.

Validation of the selectivity of Phospho-DAP12 (Tyr91) assay using Accell siRNA

THP-1 cells were plated in a 96-well plate (50,000 cells/well) and transfected with Accell siRNA DAP12, as well as a negative control. Following a 96-hour incubation, 250 µM of Pervanadate were added to the cells for 30 minutes before lysing with supplemented lysis buffer #3 (4x) for 30 minutes at room temperature under gentle shaking.

For the detection step, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-DAP12 (Tyr91) detection antibodies were added. The HTRF signal was recorded after 24 hours.

Cell transfection with siRNA DAP12 led to a significant decrease (56%) in the detection of the protein compared to the negative control (non-targeting control), demonstrating that the HTRF Phospho-DAP12 (Tyr91) assay is selective.

Assessment of phospho-DAP12 (Tyr91) levels in undifferentiated and differentiated THP-1 cells

Various cell densities of undifferentiated and differentiated THP-1 cells (treated with 150 nM PMA for 20 hours) were seeded in a 96-well microplate. The cells were then treated with 250 µM of Pervanadate for 30 minutes before being lysed with supplemented lysis buffer #3 for 30 minutes at room temperature under gentle shaking.

Next, 16 µL of the lysate were transferred into a 384-well low volume white microplate, followed by the addition of 4 µL of the HTRF Phospho-DAP12 (Tyr91) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Phospho-DAP12 (Tyr91) assay successfully detected DAP12 phosphorylation at residue Y91 in both monocytes and macrophage-like cells, with varying levels of phosphorylation.

HTRF Phospho-DAP12 (Tyr91) assay compared to Western Blot

THP-1 cells were grown in a T175 flask in complete culture medium at 37°C and 5% CO₂ until they reached 80% confluence. The cells were then treated with 250 µM of Pervanadate for 30 minutes and lysed with 3 mL of supplemented lysis buffer #3 (4x) for 30 minutes at room temperature under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer. Then 13 µL of each dilution were transferred into a low volume white microplate, followed by the addition of 3 µL of supplemented lysis buffer #3 (1x) to ensure the same quantity for both techniques. Finally, 4 µL of HTRF Phospho-DAP12 (Tyr91) detection reagents were added. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF Phospho-DAP12 (Tyr91) assay, 1000 cells/well were sufficient to detect a significant signal, while 8100 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore, under these conditions, the HTRF Phospho-DAP12 (Tyr91) assay was 8 times more sensitive than the Western Blot technique.

TREM2/DAP12 signaling pathway

The TREM2-DAP12 receptor complex plays a continuous surveillance role by scanning the neuronal microenvironment and maintaining healthy brain homeostasis in microglia cells.

DAP12 (also known as TYROBP or KARAP) is a homodimer transmembrane adaptor protein initially described as a receptor-activating subunit component of natural killer (NK) cells. It is expressed in various cell types, including monocytes, macrophages, dendritic cells, and microglia.

Due to its short extracellular domain, DAP12 itself is thought to lack ligand-binding capability. Instead, it forms complexes with ligand-binding receptors (DAP12-associated receptors) and transduces signals from these receptors into the cytoplasm.

Activation of TREM2 leads to DAP12 phosphorylation and subsequent recruitment of SYK, which promotes the phosphorylation and activation of multiple downstream mediators such as PI3K, AKT, BTK, ERK, and NFkβ.