IRF3 Antibody

What is IRF3 and why is it important in immunological research?

IRF3 is a critical transcription factor (~55 kDa) involved in the innate immune response, particularly in the detection of viral infections and the activation of type I interferons such as IFN-β. Structurally, IRF3 consists of several domains, including a DNA-binding domain, an IRF association domain, and a regulatory C-terminal domain . It remains inactive in the cytoplasm until phosphorylated following viral detection, after which it dimerizes, translocates to the nucleus, and initiates the transcription of antiviral genes . This makes IRF3 vital for studying host-pathogen interactions and antiviral immunity mechanisms.

How do I select the appropriate IRF3 antibody for my experiment?

Selection criteria should include:

• Specificity: Verify specificity through knockout validation or comparison with isotype controls. For instance, the D-3 monoclonal antibody targets amino acids 389-427 near the C-terminus of human IRF3 , while clone 3F10 recognizes amino acids 108-166 .

• Application compatibility: Ensure the antibody is validated for your specific application:

  • Western blotting (dilutions typically 1/1000-1/3000)

  • Immunoprecipitation

  • Immunofluorescence

  • Flow cytometry

  • ELISA

• Cross-reactivity profile: Some antibodies show cross-species reactivity (human, mouse, rat), while others are species-specific. For example, AF4019 shows approximately 20% cross-reactivity with recombinant mouse IRF3 in direct ELISAs .

• Conjugation status: Available as non-conjugated or conjugated with agarose, HRP, PE, FITC, or various Alexa Fluor conjugates for specialized applications .

What are the common troubleshooting issues when using IRF3 antibodies in Western blotting?

For optimal results, use freshly prepared lysates with phosphatase inhibitors to preserve IRF3 phosphorylation states. Include IRF3 knockout cell lines as negative controls to confirm band specificity, as demonstrated with HeLa parental and IRF3 knockout cell lines .

How should I optimize fixation protocols for IRF3 immunofluorescence staining?

Effective IRF3 immunofluorescence depends greatly on fixation methods:

• Fixative selection: For phosphorylated IRF3 detection, 4% paraformaldehyde is recommended (10-15 minutes at room temperature), as it better preserves protein phosphorylation compared to methanol fixation.

• Permeabilization: Use 0.1-0.5% Triton X-100 or saponin for nuclear transcription factor accessibility4. For flow cytometry applications, fixation buffers like Flow Cytometry Fixation Buffer followed by permeabilization with Flow Cytometry Permeabilization/Wash Buffer I have been validated .

• Antibody concentration: For immunofluorescence, optimal concentration ranges (e.g., 15 µg/mL for 3 hours at room temperature) should be determined experimentally .

• Signal validation: Always include appropriate controls:

  • Primary antibody omission

  • IRF3-deficient cells

  • Competing peptide blockade

Counterstaining with DAPI helps confirm nuclear translocation during IRF3 activation, while cytoplasmic staining predominates in resting cells .

What are effective strategies for detecting IRF3 activation status?

IRF3 activation can be monitored through:

• Phosphorylation-specific antibodies: Target key phosphorylation sites (Ser385/386 and Ser396), with Ser385/386 being particularly critical for IRF3 activation .

• Nuclear translocation: Use fractionation or immunofluorescence to track movement from cytoplasm to nucleus. The shift is dramatic upon viral infection or poly(I:C) stimulation.

• Dimerization assays: Non-denaturing gel electrophoresis can detect IRF3 dimers.

• Proximity ligation assay (PLA): This technique can detect IRF3 interactions with co-factors like TBK1. PLA has been used to show how Ebola virus reduces TBK1:IRF3 complexes during infection .

• Chromatin immunoprecipitation (ChIP): Quantifies IRF3 binding to target gene promoters, allowing measurement of binding intensity at sites like IFN-β promoter.

How can I validate the specificity of IRF3 antibodies for my research application?

Rigorous validation approaches include:

• Genetic controls:

  • Use IRF3 knockout cell lines (as demonstrated in Western blot validations with HeLa parental and IRF3 knockout cell lines)

  • siRNA/shRNA knockdown samples

• Peptide competition: Pre-incubate antibody with immunizing peptide to confirm binding specificity.

• Cross-validation: Test multiple antibodies targeting different IRF3 epitopes.

• Stimulation experiments: Verify increased signal upon appropriate stimulation (viral infection, poly(I:C)).

• Multiple techniques: Confirm findings using complementary methods (IF, WB, IP).

How can IRF3 antibodies be used to investigate cross-talk between different IRF family members?

IRF family cross-talk investigation requires specialized approaches:

• Sequential ChIP (ChIP-reChIP): This technique helps identify co-occupancy of IRF3 with other IRFs (IRF5, IRF9) at the same genomic region. Studies have shown that IRF3, IRF5, and IRF9 regulate overlapping but distinct sets of target genes .

• Co-immunoprecipitation with specificity controls: When investigating interactions, use antibodies targeting unique regions to prevent cross-reactivity between family members. This is critical as IRFs share significant homology in DNA-binding domains.

• Isoform-specific detection: Ensure antibody specificity for detecting the alpha isoform without cross-reactivity to beta isoforms, as demonstrated with GSK3 isoform-specific antibodies4.

• Combined stimulation experiments: Challenge cells with multiple stimuli to observe competition or cooperation between IRF pathways. Research has shown that different ligand combinations can either inhibit or enhance IRF3, IRF5, and IRF9 binding to their respective genomic regions .

What are the best practices for using IRF3 antibodies to study viral evasion mechanisms?

Viral evasion studies require specialized experimental approaches:

• Subcellular fractionation: Determine if viruses alter normal IRF3 distribution patterns. For example, Ebola virus has been shown to sequester IRF3 in viral inclusion bodies (IBs) .

• Time-course experiments: Track IRF3 phosphorylation, dimerization, and nuclear translocation kinetics during infection.

• Co-localization studies: Use dual-labeling with viral proteins and IRF3. This approach revealed how EBOV traps IRF3 in specific cellular compartments.

• Proximity ligation assay (PLA): Detect interactions between IRF3 and viral proteins or usual binding partners. PLA experiments demonstrated that EBOV infection significantly reduces TBK1:IRF3 complexes that normally form after poly(I:C) stimulation .

• Functional rescue experiments: Determine if supplementing IRF3 activity can overcome viral evasion strategies.

How can phospho-specific IRF3 antibodies be leveraged to dissect signaling pathways?

Phospho-specific antibodies enable precise pathway interrogation:

• Site-specific analysis: Different phosphorylation sites have distinct functional impacts. Research has demonstrated that mutations at Ser-385/386 abolish promotion of IFN-β, ISG15, and IFITM3 expression, while Ser-396 mutations only moderately affect these pathways .

• Temporal regulation: Track phosphorylation sequence during activation using site-specific antibodies.

• Pathway inhibitor studies: Combined with kinase inhibitors to map phosphorylation dependencies.

• Quantitative analysis: Use phospho-to-total IRF3 ratios for accurate activation measurement.

• Mutant complementation: Compare antibody reactivity in cells expressing wild-type versus phosphorylation site mutants of IRF3. Studies confirmed that both S385/386A and S396A mutations strongly diminished ISG15 and IFITM3 protein expression .

How should IRF3 antibodies be stored and handled to maintain optimal performance?

Proper handling ensures antibody longevity and consistent results:

• Storage temperature: Most IRF3 antibodies should be stored at 2-8°C and should not be frozen . For long-term storage, small aliquots at -20°C may be recommended to prevent freeze-thaw cycles.

• Buffer considerations: Many IRF3 antibodies come in phosphate-buffered saline (PBS) with preservatives like 0.02% sodium azide and 10% glycerol .

• Light sensitivity: For fluorophore-conjugated antibodies (e.g., Alexa Fluor 488-conjugated IRF3 antibodies), protection from light is essential .

• Shelf life: Typical guarantee period is 12 months from date of despatch when stored properly .

• Working solution preparation: Dilute only the amount needed for immediate use in appropriate buffer.

What are the key differences between using IRF3 antibodies in flow cytometry versus immunofluorescence microscopy?

For flow cytometry, successful detection of IRF3 has been demonstrated using Alexa Fluor 488-conjugated monoclonal antibodies with specific fixation and permeabilization buffers in cell lines like Daudi human Burkitt's lymphoma . For immunofluorescence, NorthernLights 557-conjugated secondary antibodies have been used successfully with primary IRF3 antibodies at 15 µg/mL concentration for detailed subcellular localization .

How can IRF3 antibodies be effectively used in ChIP-seq experiments to study genome-wide binding patterns?

For successful IRF3 ChIP-seq:

• Crosslinking optimization: Typically 1% formaldehyde for 10 minutes, but may require titration.

• Antibody selection: Choose ChIP-validated antibodies targeting the DNA-binding domain or C-terminal region. Avoid phospho-specific antibodies unless specifically studying activated IRF3.

• Chromatin fragmentation: Aim for 200-500bp fragments for optimal resolution of binding sites.

• Controls: Include:

  • Input chromatin

  • IgG control

  • IRF3-deficient cells

  • Stimulated vs. unstimulated samples to capture activation-dependent binding

• Data analysis: Focus on motif analysis to identify IRF3 binding elements (IBEs) and co-occurring transcription factor motifs. Research has shown that IRF3 binding co-occurs frequently with RelA binding sites .

• Validation: Confirm key peaks with ChIP-qPCR, especially at known IRF3 target genes like IFN-β, CCL5, and CXCL10 .

How can IRF3 antibodies be used to study viral pathogenesis mechanisms?

IRF3 antibodies provide critical insights into viral pathogenesis:

• Evasion mechanism profiling: Different viruses employ distinct strategies to counteract IRF3. For example, Ebola virus sequesters IRF3 in viral inclusion bodies to prevent it from activating interferon responses .

• Comparative analysis: Study how various viral proteins (NS1, NS3/4A, VP35) target different aspects of IRF3 activation.

• Time-course studies: Track IRF3 phosphorylation, dimerization, and nuclear translocation during different phases of viral infection.

• Mutant virus comparisons: Compare wild-type viruses with mutants lacking specific immune evasion genes.

• Host-range determination: Investigate how viral antagonism of IRF3 varies across host species, particularly at host-species barriers.

What approaches can be used to study IRF3 signaling in primary immune cells versus cell lines?

Primary cell research requires specific considerations:

• Antibody validation: Revalidate antibodies in primary cells, as expression levels and isoforms may differ from cell lines:

  Cell Type	Recommended Antibody Dilution (WB)	Optimization Notes
  Cell lines (HeLa, U937)	1:1000-3000	Standard protocols usually effective
  Primary human MDMs	1:500-1000	May require longer exposure times
  Mouse BMDMs	1:500-1000	May show species-specific differences
  Human PBMCs	1:500-1000	Higher background common

• Transfection challenges: For primary cells resistant to transfection, antibody-based detection becomes even more critical for studying endogenous IRF3.

• Activation kinetics: Primary cells often show different IRF3 activation kinetics than immortalized lines. Design time-course experiments accordingly.

• Cell-type specific interactions: Investigate co-binding with lineage-specific transcription factors, as IRF3 frequently collaborates with other transcription factors like AP-1 and NF-κB .

• Ex vivo validation: Confirm key findings from cell cultures in tissue samples using IHC with IRF3 antibodies.

How are IRF3 antibodies being used to investigate the interplay between IRF3 and co-regulatory proteins?

Recent research focuses on complex interactions:

• Co-immunoprecipitation coupled with mass spectrometry: Identify novel IRF3-interacting proteins using antibodies that don't disrupt protein complexes.

• Proximity labeling techniques: BioID or APEX2 fusions with IRF3 coupled with specific antibody detection.

• Sequential ChIP (ChIP-reChIP): Investigate co-occupancy of IRF3 with other transcription factors at genomic loci, revealing extensive collaboration between IRF3 and RelA in antiviral responses .

• FRET/BRET analysis: Study real-time interactions between IRF3 and partners using antibody-based detection systems.

• PBLD interactions: Recent findings show that phenazine biosynthesis-like domain-containing protein (PBLD) enhances type I interferon expression through IRF3, suggesting new regulatory mechanisms .

What are the latest techniques for studying post-translational modifications of IRF3 using specific antibodies?

Advanced PTM research approaches include:

• Phospho-specific antibodies: Critical for distinguishing between various activated forms. Research has shown differential importance of phosphorylation sites, with Ser-385/386 being more critical than Ser-396 for certain pathways .

• Mass spectrometry validation: Confirm antibody-detected modifications with MS analysis of immunoprecipitated IRF3.

• High-resolution imaging: Study spatiotemporal dynamics of IRF3 modifications in different cellular compartments.

• Single-cell analysis: Detect heterogeneity in IRF3 activation states within populations using phospho-flow cytometry.

• Orthogonal labeling strategies: Combine antibody detection with chemical labeling of specific modifications.

How can IRF3 antibodies contribute to therapeutic research targeting innate immune pathways?

Therapeutic applications leverage IRF3 antibodies in multiple ways:

• Compound screening: Use phospho-IRF3 antibodies to screen for modulators of innate immune signaling.

• Target validation: Confirm mechanism of action for compounds designed to modulate IRF3 activity.

• Biomarker development: Explore IRF3 activation status as a predictive or pharmacodynamic biomarker.

• Safety assessment: Evaluate potential off-target effects of therapeutics on innate immune pathways.

• Antibody-based therapies: Development of antibodies that could potentially deliver cargoes to cells with aberrant IRF3 activity.

• Viral evasion countermeasures: Research into preventing viral sequestration of IRF3, such as that seen with Ebola virus, could lead to novel broad-spectrum antiviral approaches .