Interferon regulatory factor 5 (IRF5) is a transcription factor involved in innate immunity and inflammatory responses, regulating the expression of type I interferons (IFN-alpha and IFN-beta) and pro-inflammatory cytokines. Activated by pattern recognition receptors such as Toll-like receptors (TLR), MDA5, or STING in response to viral infection, IRF5 plays a crucial role in antiviral defense. In its inactive form, IRF5 resides in the cytoplasm of uninfected cells. Upon activation, it undergoes phosphorylation, dimerization, and nuclear localization to drive gene expression. Dysregulation of IRF5 is linked to autoimmune diseases such as lupus and rheumatoid arthritis, as well as inflammatory cancers, where it modulates tumor-associated macrophages.
The AlphaLISA SureFire Biotin-Free Human Phospho-IRF5 (Ser446) Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-IRF5 (Ser446) in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho-AlphaLISA SureFire Biotin Free assay principle
The Phospho-AlphaLISA SureFire Biotin Free assay measures a protein target when phosphorylated at a specific residue in a biological sample (e.g. cell lysate).
The assay uses two antibodies which recognize the phospho epitope and a distal epitope on the target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure™ agent to specifically immobilize the assay specific antibody, labeled with a CaptSure tag. Donor Beads are coated with a proprietary CaptSure3 agent to capture one of the detection antibodies, which is labeled with CaptSure3 tag. In the presence of phosphorylated protein, the two antibodies bring the Donor and Acceptor Beads in close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphoprotein present in the sample.
Phospho-AlphaLISA SureFire Biotin Free two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho-AlphaLISA SureFire Biotin Free detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho-AlphaLISA SureFire Biotin Free one-plate assay protocol
Detection of Phosphorylated target protein with AlphaLISA SureFire Biotin Free reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire quality.
Induction of IRF5 Phosphorylation in CpG-B treated endogenous cell lines
RPMI 8226 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of CpG-B ODN 2006 or ODN 1668 (MedChem Express, HY-150218, HY-150726 respectively) for 3 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, supernatant was removed, and cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Aggregate, Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, CpG-B triggered a dose-dependent increase in Aggregate and Phospho (Ser446) IRF5 while Total levels remained unchanged.
Daudi cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of CpG-B ODN 2006 (MedChem Express, HY-150218) for 3 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, supernatant was removed, and cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 80,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
As expected, CpG-B triggered a dose-dependent increase in Phospho (Ser446) IRF5 while Total levels remained unchanged.
Activation of IRF5 Phosphorylation in R848 treated cells
RPMI 8226 cells were seeded in a 96-well plate (400,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of R848 (InvivoGen) for 4 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, supernatant was removed and cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 80,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Treatment with a TLR7/8 agonist, R848, induced a dose-dependent increase in the levels of Phospho (Ser446) IRF5 while Total levels remained unchanged.
Increase of Phospho IRF5 (Ser446) in Calyculin A treated cells
THP-1 cells were seeded in a 96-well plate (200,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were treated with increasing concentrations of Calyculin A for 3 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, supernatant was removed, and cells were lysed with 50 µL of Lysis Buffer for 10 minutes at RT with shaking (350 rpm). Aggregate, Phospho (Ser446) and Total IRF5 levels were evaluated using respective AlphaLISA SureFire Biotin Free assays. For the detection step, 10 µL of cell lysate (approximately 40,000 cells) was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at RT. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
Calyculin A treatment triggered a dose-dependent increase in the levels of Phospho (Ser446) and Aggregate IRF5 while Total levels remained unchanged.
