Human and Mouse Phospho SYK (Tyr525/526) Biotin Free 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. Ramos cells were seeded at 200K cells/mL in a 96-well plate in serum free RPMI 1640 medium and treated with Anti-IgM antibody at the indicated concentrations for 20 minutes. Cells were lysed with the addition of 5X Lysis Buffer and assayed separately for Phospho (Tyr525/526) and Total SYK using respective SureFire® Biotin Free kits. Equivalent to approximately 8,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were treated with 35 ng/mL TGF-β for 18 hours. Cells were seeded at 400K cells/well in a 96-well plate in serum free RPMI 1640 medium and treated with 20 μM TREM2 Activator (MedChemExpress, HY-148095) for 2 minutes. Cells were lysed with the addition of 5X Lysis Buffer and assayed separately for Phospho (Tyr525/526) and Total SYK using respective SureFire® Biotin Free kits. Equivalent to approximately 16,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. U937 cells were seeded at 400K cells/well in a 96-well plate in RPMI 1640 medium and treated with Pervanadate at the indicated concentrations for 30 minutes. Cells were lysed with the addition of 5X Lysis Buffer and assayed separately for Phospho (Tyr525/526) and Total SYK using respective SureFire® Biotin Free kits. Equivalent to approximately 16,000 cells/datapoint.