IRF7 Total 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 200K cells/well and treated with IFNα or IFNβ at the indicated concentrations for 6 hours. Cells were washed with HBSS and lysed with Lysis Buffer. Lysates were assayed separately for Total IRF7 and Cofilin Total using respective SureFire® Ultra™ kits. Equivalent to approximately 20,000 cells/datapoint for Total IRF7 and 400 cells/datapoint for Total Cofilin.

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 IFNα or IFNβ at the indicated concentrations for 24 hours. Cells were washed with HBSS, lysed with Lysis Buffer and assayed separately Total IRF7 and Cofilin Total using respective SureFire® Ultra™ kits. Equivalent to approximately 1,000 cells for Total IRF7 and 40 cells for Cofilin Total.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded at 400K cells/well 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 Total IRF7 using the SureFire® Ultra™ kit. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded at 400K cells/well 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™ kits. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. THP-1 cells were seeded at 100K cells/well and incubated in medium containing 100nM PMA for 24 hours. Cells 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™ kits. Equivalent to approximately 10,000 cells/datapoint.

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

Data obtained from measurement of Total IRF7 protein levels in various cell types. Adherent cell lines were seeded at 40K cells/well and incubated overnight and lysed with Lysis Buffer. Suspension cell lines were seeded at 400K cells/well and lysed with Lysis Buffer. Suspension and adherent cell lysates were then assayed for Total IRF7 using the SureFire® Ultra™ kit. Equivalent to approximately 4,000 cells/datapoint for adherent and 40,000 cells/datapoint for suspension cell lines.