IRF7 Phospho (Ser477) Assay Kit Human

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. THP-1 cells were seeded at 400K cells/well in complete medium and treated with IFNα or IFNβ at the indicated concentrations for 6 hours. Cells were washed with HBS and lysed with Lysis Buffer. Lysates were assayed separately for Phospho (Ser477) IRF7 and Cofilin Total using respective SureFire® Ultra™ assay kits. Equivalent to approximately 80,000 cells/datapoint for Phospho (Ser477) IRF7 and 8,000 cells/datapoint for Cofilin Total.

Data obtained with a 2-plate, 2-incubated protocol. THP-1 cells were seeded at 100K cells/well in medium containing 100nM PMA and incubated for 24 hours. THP-1 derived macrophages were then treated with 250 ng/mL of IFNα or IFNβ for a further 24 hours. Cells were washed with HBSS, lysed with Lysis Buffer and assayed separately for Phospho (Ser477) and Total IRF7 using respective SureFire® Ultra™ assay kits. Equivalent to approximately 10,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubated protocol. HT 29 cells were seeded at 20K cells/well and incubated overnight. Cells were treated with 100 ng/mL of IFNα or IFNβ for 24 hours. Cells were washed with HBSS, lysed with Lysis Buffer and assayed for Phospho (Ser477) IRF7 using the SureFire® Ultra™ assay kit. Equivalent to approximately 2,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubated protocol. THP-1 cells were seeded at 400K cells/well in complete medium and treated with 20 μM of STING agonist, diABZI at the indicated time points. Cells were washed with HBSS, lysed with Lysis Buffer and assayed for Phospho (Ser477) IRF7 using the SureFire® Ultra™ assay kit. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubated protocol. THP-1 cells were seeded at 400K cells/well in complete medium and treated with 100 μg/mL of STING ligand, 2’3’ cGAMP for 4 hours. Cells were washed with HBSS, lysed with Lysis Buffer and assayed separately for Phospho (Ser477) and Total IRF7 using respective SureFire® Ultra™ assay kits. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubated protocol. THP-1 cells were seeded at 100K cells/well in medium containing 100 nM PMA and incubated for 18 hours. THP-1 derived macrophages were then treated with Calyculin A at the indicated concentrations for 30 minutes. Cells were washed with HBSS, lysed with Lysis Buffer and assayed for Phospho (Ser477) and Total IRF7 using respective SureFire® Ultra™ assay kits. Equivalent to approximately 10,000 cells/datapoint.