Human Phospho-DAP12 (Tyr91) Detection Kit

The AlphaLISA™ SureFire® Ultra™ assay enables the rapid and sensitive detection of total and phosphorylated cellular proteins. AlphaLISA™ assays utilize two bead types: Acceptor Beads and Donor Beads. The Acceptor Bead is coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody which is labeled with a CaptSure™ tag. The Donor Bead is coated with streptavidin to capture the biotinylated antibody.  

In the presence of a target protein, the two target-specific antibodies bring Donor and Acceptor Beads into close proximity. When the Donor Beads are activated by a laser (680 nm), singlet oxygen is transferred to the Acceptor Bead leading to the production of an Alpha signal. The amount of light emission (615 nm) from the Acceptor Bead is directly proportional to the amount of target protein present in the sample. If an Acceptor Bead is not in close proximity (i.e. 200 nm) of a Donor bead, little to no signal is produced over background. 

The assay can be executed in a 1-plate or 2-plate assay protocol (Refer to Manual for more details).  

1-plate assay protocol: culturing of cells, treatment, lysis and assay are performed in a single well, enabling miniaturization in high throughput screening programs. 

2-plate assay protocol: cells are cultured and treated in a 96-well culture plate and lysates are then transferred into a separate plate for assay. This format allows the evaluation of multiple targets from a single lysate. 

Data obtained with a 2-plate, 2-incubation protocol. THP1 cells were prepared at 600K/mL in 10% FBS containing media and treated with 35 ng/mL TGFb for 20 hours. Cells were then washed with HBSS + 0.1% BSA and seeded at 400K cells/well in a 96 well plate. Cells were treated with a TREM2 Activator at the indicated concentrations for 10 minutes and lysed with 5X Lysis Buffer. Assayed separately for Phospho (Tyr91) and Total DAP12, and Phospho SYK (Tyr525/526) using respective SureFire® Ultra™ kits. Equivalent to approximately 16,000 cells or 1,600 cells/datapoint (DAP12 Total).

Data obtained with a 2-plate, 2-incubation protocol. THP1 Wild Type cells (WT) and THP1-DAP12 Knockout cells (KO) were harvested at 800K/mL and washed in HBSS + 0.1% BSA. Cells were seeded at 400K cells/well in a 96 well plate and incubated with Concanavalin A at the indicated concentrations for 10 minutes. Cells were spun down, washed in HBSS and lysed in Lysis Buffer. Assayed separately for Phospho (Tyr91), Total DAP12 and Total TREM2 using respective SureFire® Ultra™ kits. Equivalent to approximately 20,000 cells/datapoint (Phospho DAP12 Tyr91) or 2,000 cells/datapoint (Total DAP12 and Total TREM2).