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HTRF Human IRF5 Detection kit HTRF®

The HTRF Human IRF5 Detection kit is designed to detect the expression level of IRF5 in cell lysates.

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  • All inclusive kit All inclusive kit
  • No-wash No-wash
  • Low sample consumption Low sample consumption

The HTRF Human IRF5 Detection kit is designed to detect the expression level of IRF5 in cell lysates.

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Overview

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.

Benefits

  • SPECIFICITY
  • PRECISION

HTRF Human IRF5 assay principle

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.

Principle of the HTRF Human IRF5 assay

HTRF Human IRF5 two-plate assay protocol

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.

2-plate protocol of the HTRF Human IRF5 assay

Human IRF5 assay validation in different cellular densities of THP-1 cells

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.

Validation of the HTRF Human irf5 assay in thp-1 cells

IRF5 simplified pathway

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.

Simplified pathway of IRF5

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Product Insert IRF5 Kit / 64HIRF5PEG-64HIRF5PEH

64HIRF5PEG-64HIRF5PEH - Product Insert

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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