IRF5 Total Biotin Free Assay Kit Human and Mouse

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. RPMI 8226 cells were seeded at 200K/well in a 96-well plate. Cells were then treated with CpG-B ODN 2006 at the indicated concentrations for 3 hours. Cells were spun down at 1200 RPM for 5 minutes, supernatant was removed and cells were lysed with Lysis Buffer and assayed separately for Aggregate, Phospho (Ser446) and Total IRF5 using respective SureFire® Biotin Free kits. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. RPMI 8226 cells were seeded at 200K/well in a 96-well plate. Cells were then treated with CpG-B ODN 1668 at the indicated concentrations for 3 hours. Cells were spun down at 1200 RPM for 5 minutes, supernatant was removed and cells were lysed with Lysis Buffer and assayed separately for Aggregate, Phospho (Ser446) and Total IRF5 using respective SureFire® Biotin Free kits. Equivalent to approximately 40,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. RPMI 8226 cells were seeded at 400K/well in a 96-well plate. Cells were then treated with R848 at the indicated concentrations for 4 hours. Cells were spun down at 1200 RPM for 5 minutes, supernatant was removed and cells were lysed with Lysis Buffer and assayed separately for Phospho (Ser446) and Total IRF5 using respective SureFire® Biotin Free kits. Equivalent to approximately 80,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. Daudi cells were seeded at 400K/well in a 96-well plate. Cells were then treated with CpG-B ODN 2006 at the indicated concentrations for 3 hours. Cells were spun down at 1200 RPM for 5 minutes, supernatant was removed and cells were lysed with Lysis Buffer and assayed separately for Phospho (Ser446) and Total IRF5 using respective SureFire® Biotin Free kits. Equivalent to approximately 80,000 cells/datapoint.

A549 wild type (WT) and A549 IRF5 knockout (KO) (Abcam, ab301006) cells were cultured to confluency in T75 flasks. Each flask was lysed in 2 mL of Lysis Buffer and lysates were evaluated for Total IRF5 using the SureFire® Biotin Free kit. Equivalent to approximately 10,000 cells/datapoint.

Data obtained with a 2-plate, 2-incubation protocol. Adherent cell lines were seeded at 40K/well in a 96-well plate and incubated overnight. Suspension cell lines were seeded at 400K/well in HBSS + 0.1% BSA in a 96-well plate. Adherent and suspension cell lines were then lysed with Lysis Buffer, lysates were further diluted to 50% and evaluated for Total IRF5 using the SureFire® Biotin Free kit. Equivalent to approximately 2,000 cells/datapoint for adherent cell lines and 20,000 cells/datapoint for suspension cell lines.