Human IFN alpha kit
No-wash kit to quantify released Human IFN alpha
The HTRF Human IRF5 Detection kit is designed to detect the expression level of IRF5 in cell lysates.
The Human HTRF IRF5 detection kit conveniently and accurately quantifies IRF5 in cell lysates. In virus-infected cells, phosphorylation of transcription factor IRF5 induces the production of antiviral and proinflammatory cytokines, key elements in the innate immunity system. This IRF5 assay can be used from basic research through to preclinical drug discovery phases, and contains everything you need. It offers increased throughput compared to ELISA/WB.
The HTRF Human IRF5 assay quantifies the expression level of human IRF5 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. In presence of IRF5 in a cell lysate, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of human IRF5 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
THP-1 cells were plated at 100,000 cell/well under 50 µl in a 96-well plate in complete culture medium. For untreated cells, 10 µl of completed culture medium was added. For siRNA treated samples, 10 µl containing a mix of Lipofectamine® RNAiMax/siRNA for IRF5 was added. Cells were then incubated for 24h or 48h at 37°C, 5% CO2.
After incubation, cells were lysed with 20 µL of supplemented lysis buffer #2 at 4X for 30 minutes at RT under gentle shaking.
16 µL of lysate were transferred into a low volume white microplate before the addition of 2 µL of the HTRF d2 detection reagent and 2 µL HTRF Eu-K detection reagent. The HTRF signal was recorded after ON incubation.
IRF5 is a key transcriptional regulator of type I interferon (IFN-alpha and IFN-beta)-dependent immune responses. It plays a critical role in the innate immune response against DNA and RNA viruses. Found in an inactive form in the cytoplasm of uninfected cells, IRF3 becomes overexpressed and phosphorylated by various mediators following viral infection sensitive pathways. Signaling from Tol-like receptors (TLR), MDA5, or STING can all contribute to its expression and activation. This induces a conformational change, leading to its dimerization and nuclear localization, where it can activate type I IFN gene expression.
Physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
In collaboration with Bayer - Scientific Presentations
A fun video introducing you to phosphorylation assays with HTRF - Videos
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes
Learn how to reduce time and sample consumption - Application Notes
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