Human TREM2 / DAP12 Complex Total 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 medium and treated with 35 ng/mL TGFβ for 20 hours. Cells were 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. Lysates were assayed separately for TREM2/DAP12 Complex, TREM2 Total and DAP12 Total using respective SureFire® Ultra™ kits. Equivalent to approximately 16,000 cells/datapoint (TREM2/DAP12 Complex) or 1,600 cells/datapoint (TREM2 and DAP12 Total).

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 TGFβ for 20 hours. Cells were washed with HBSS + 0.1% BSA and seeded at 400K cells/well in a 96 well plate. Cells were treated with 10 μM TREM2 Activator for the indicated time points, lysed with 5X Lysis Buffer and assayed for TREM2/DAP12 Complex. Equivalent to approximately 16,000 cells/datapoint.

Control lysate prepared from THP1 cells was serially diluted in Lysis buffer and assayed for TREM2/DAP12 Complex. Approximate number of cells/datapoint is indicated.