Description
Interferon regulator factor 5, IRF5, is a transcriptional regulator of type I interferon (IFN-alpha and IFN-beta)-dependent immune responses. This transcription factor plays a key role in the innate immune response against DNA and RNA viruses. IRF5 is present in the cytoplasm of uninfected cells in an inactive form. Various mediators of the pathways that follow viral infection can overexpress and phosphorylate IRF5. IRF5 is associated with various cancers and autoimmune diseases.
The AlphaLISA SureFire Ultra Human IRF5 Aggregate Detection Kit is a sandwich immunoassay for the quantitative detection of aggregate IRF5 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
AlphaLISA SureFire Ultra protein dimer / aggregate detection assay principle
The AlphaLISA SureFire Ultra dimer/aggregate assay measures the levels of a given protein in a dimeric, multimeric or any other aggregated form in a biological sample (e.g. cell lysate).
The assay uses a single antibody clone tagged with both CaptSure™ peptide and biotin, that recognizes exposed epitopes on the multivalent 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 antibody labeled with a CaptSure tag. Donor Beads are coated with streptavidin to capture the same specific antibody, which is biotinylated. In the presence of the multivalent 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 multimeric protein present in the sample.
AlphaLISA SureFire Ultra dimer / aggregate detection 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 dimer or aggregate AlphaLISA SureFire Ultra 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.
AlphaLISA SureFire Ultra protein dimer / aggregate detection one-plate assay protocol
Detection of the dimer or aggregate with AlphaLISA SureFire Ultra 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 Ultra quality.
Increase of IRF5 Aggregate in CpG-B ODN-2006 treated cells
RPMI 8226 cells were seeded in a 96-well plate (500,000 cells/well) in complete medium and incubated for approximately 10 minutes at 37°C, 5% CO2. The cells were then treated with increasing concentrations of CpG-B ODN 2006 for 3 hours.
After treatment, the cells were spun down at 1200 rpm for 5 minutes, washed with HBSS + 0.1% BSA and 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 Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 cells) were 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 ODN 2006 triggered a dose-dependent increase in the levels of Aggregate and Phospho (Ser446) IRF5 while Total IRF5 levels remained unchanged.
Increase of IRF5 Aggregate 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, washed with HBSS + 0.1% BSA and 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 Ultra assays. For the detection step, 10 µL of cell lysate (approximately 50,000 cells) were 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, Calyculin A triggered a dose-dependent increase in the levels of Aggregate and Phospho (Ser446) IRF5 while Total IRF5 levels remained unchanged.
