DAP12, also known as TYRO protein tyrosine kinase-binding protein, functions as a signaling adapter for a number of innate immune system cell surface receptors. By activating SYK or Zap-70 and other downstream targets, crosslinking receptors linked to DAP12 causes tyrosine residues in their cytoplasmic ITAM to be phosphorylated by SRC family kinases, which initiates downstream signaling. By monitoring the neuronal surroundings and preserving a healthy brain homeostasis in microglia cells, the TREM2-DAP12 receptor complex seems to play an ongoing role.
The AlphaLISA SureFire Human Phospho-DAP12 (Tyr91) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-DAP12 (Tyr91) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
Alpha SureFire kits can be used for:
Cellular kinase assays
Receptor activation studies
Screening
Phospho-AlphaLISA SureFire Ultra assay principle
The Phospho-AlphaLISA SureFire Ultra assay measures a target protein when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture one of the detection antibodies, which is biotinylated. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
TREM2 Activator rapidly induces DAP12 phosphorylation in THP-1 and primary macrophages
THP-1 cells were treated with 35 ng/mL TGFb for 18 hours at 37°C, 5% CO2. Cells were harvested and seeded in a 96-well plate (400,000 cells/well) in HBSS + 0.1% BSA and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.
After treatment, cells were lysed with the addition of 50 µL of 5X Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DAP12 Phospho (Tyr91) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 16,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.
TREM2 activator induced DAP12 phosphorylation within just 2 minutes, peaking after 10 minutes of treatment.
PBMCs were isolated from healthy donors and cultured for 6 days in complete DMEM containing 20 ng/mL M-CSF to differentiate them into macrophages. Macrophages were seeded in a 96-well plate (40,000 cells/well) in complete DMEM, and incubated overnight at 37°C, 5% CO2. Cells were treated with 35 ng/mL TGFb for 18 hours and then treated with 20 µM TREM2 Activator (MedChemExpress, Cat # HY-148095) for various time points.
After treatment, cells were lysed in 200 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). DAP12 Phospho (Tyr91) levels were evaluated by AlphaLISA SureFire Ultra. For the detection step, 10 µL of cell lysate (approximately 2,000 cells) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark.
As expected, TREM2 activator induced Phospho DAP12 (Tyr91) very quickly, peaking after 10 minutes of treatment.
Concanavalin A induces DAP12 phosphorylation in THP-1 cells
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in HBSS + 0.1% BSA and treated with increasing concentrations of Concanavalin A for 10 minutes.
After treatment, the cells were spun down at 300 RCF for 5 minutes, treatment removed and then lysed with 100 µL Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Tyr91) and Total DAP12 levels were evaluated using respective AlphaLISA SureFire Ultra assays. For the detection step, 10 µL of cell lysate (approximately 20,000 cells for Phospho or 2,000 cells for Total) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, Concanavalin A triggered a dose-dependent increase in the levels of Phospho DAP12 (Tyr91) with no significant changes in Total DAP12.
