
Feature | Specification |
---|---|
Application | Cell Signaling |
Protocol Time | 2h at RT |
Sample Volume | 10 µL |
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 IRF5 Aggregate Detection Kit is a sandwich immunoassay for the quantitative detection of IRF5 aggregate in cellular lysates, using Alpha Technology.
Formats:
AlphaLISA SureFire Ultra kits are compatible with:
AlphaLISA SureFire Ultra kits can be used for:
The Aggregation-AlphaLISA SureFire Biotin Free assay uses a single antibody which recognizes an exposed epitope on the aggregated target protein, that is conjugated with either a CaptSure™ tag or with CaptSure3 tag. 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 the CaptSure tag. Donor beads are coated with a proprietary CaptSure3 agent to capture the antibody labeled with the CaptSure3 tag. In the presence of aggregated target protein, the Donor and Acceptor beads are brought into close proximity whereby the singlet oxygen transfers energy to excite the Acceptor bead, allowing the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of aggregated protein present in the sample.
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 Aggregation-AlphaLISA SureFire Biotin Free detection reagents. This protocol allows for the cells viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Detection of aggregated 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 Ultra quality.
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.
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 2.5 µg/mL 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). Aggregate and Phospho (Ser446) 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 R848 caused a modest increase in IRF5 Aggregate and increased levels of Phospho (Ser446) IRF5. Total IRF5 levels remained unchanged, data not shown.
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) were transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hours 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.
Application |
Cell Signaling
|
---|---|
Automation Compatible |
Yes
|
Brand |
AlphaLISA SureFire Biotin-Free
|
Detection Modality |
Alpha
|
Protocol Time |
2h at RT
|
Sample Volume |
10 µL
|
Shipping Conditions |
Shipped in Blue Ice
|
Target |
IRF5
|
Target Class |
Phosphoproteins
|
Target Species |
Human
|
Technology |
Alpha
|
Therapeutic Area |
Inflammation
|
Unit Size |
500 assay points
|
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