FAF3 Antibody

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

IRF3 (Interferon Regulatory Factor 3) is a 60 kDa member of the IRF family of proteins that plays a critical role in innate immune responses, particularly against viral infections. IRF3 contains one DNA binding domain (amino acids 7-107), a nuclear export signal (amino acids 139-149), and multiple phosphorylation sites (amino acids 395-407). Following viral infection, IRF3 undergoes phosphorylation, which triggers its nuclear translocation and stimulates interferon production . The importance of IRF3 in immunological research stems from its central role in antiviral defense mechanisms and its involvement in multiple signaling pathways including the cGAS-STING pathway, which has been shown to inhibit TLR9-mediated IFN production in human plasmacytoid dendritic cells .

What are the common applications for IRF3 antibodies in basic research?

IRF3 antibodies are employed in several fundamental research applications:

• Flow cytometry: Human IRF3 Alexa Fluor® 488-conjugated antibodies are effectively used to detect IRF3 in various cell types, including Daudi human Burkitt's lymphoma cell lines. This application requires proper fixation and permeabilization of cells .

• Western blotting: IRF3 antibodies can detect human IRF3 in Western blots, allowing researchers to quantify protein expression levels and phosphorylation states .

• Immunofluorescence microscopy: For visualizing cellular localization of IRF3, particularly its translocation from cytoplasm to nucleus during activation.

• Immunoprecipitation: To isolate IRF3 and its binding partners for interaction studies.

• ChIP assays: For studying IRF3 binding to target gene promoters.

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

For optimal performance of IRF3 antibodies, particularly the Human IRF3 Alexa Fluor® 488-conjugated Antibody, researchers should follow these evidence-based guidelines:

• Storage temperature: Maintain at 2 to 8°C for up to 12 months from date of receipt as supplied .

• Light protection: Always protect fluorophore-conjugated antibodies from light to prevent photobleaching .

• Avoid freezing: Do not freeze conjugated antibodies as this can damage the fluorophore and reduce antibody performance .

• Aliquoting: For frequent use, consider preparing small aliquots to minimize freeze-thaw cycles.

• Buffer conditions: Maintain recommended buffer conditions as specified in product documentation.

• Reconstitution: Follow manufacturer's guidelines for reconstitution of lyophilized antibodies.

• Working dilutions: Prepare fresh working dilutions on the day of the experiment.

How can I optimize intracellular staining protocols for IRF3 detection by flow cytometry?

Optimizing intracellular staining for IRF3 requires careful attention to several parameters:

• Fixation optimization: Use Flow Cytometry Fixation Buffer to effectively preserve cellular architecture while maintaining antibody epitope accessibility .

• Permeabilization protocol: Employ Flow Cytometry Permeabilization/Wash Buffer I to ensure antibody access to intracellular IRF3 .

• Antibody titration: Perform a titration series (0.1-10 μg/ml) to determine optimal antibody concentration for your specific cell type.

• Stimulation conditions: For phospho-IRF3 detection, optimize viral stimulation or pathway activator concentrations and time points.

• Controls:

  • Include isotype control antibody (e.g., Catalog # IC0041G) to assess non-specific binding

  • Use IRF3-knockout or IRF3-silenced cells as negative controls

  • Include positive controls such as virus-infected cells with known IRF3 activation

• Co-staining considerations: When combining with other antibodies, verify that fluorophores have minimal spectral overlap or perform appropriate compensation.

• Incubation parameters: Optimize temperature, duration, and buffer composition for your specific experimental conditions.

What are the key considerations when comparing IRF3 antibodies recognizing different epitopes?

When selecting and comparing IRF3 antibodies recognizing different epitopes, researchers should consider:

• Epitope location: The Human IRF3 Alexa Fluor® 488-conjugated Antibody recognizes amino acids 206-427 of recombinant human IRF3 . Consider whether your experiment requires detection of:

  • DNA binding domain (aa 7-107)

  • Nuclear export signal region (aa 139-149)

  • Phosphorylation sites (aa 395-407)

• Alternative splice form detection: Be aware that IRF3 has multiple splice variants, including:

  • A variant with deletion of aa 201-327

  • A variant with the same deletion plus an alternate start site at Met147

  • A variant with a 125 aa substitution for the C-terminal 100 aa (aa 328-427)

• Phosphorylation-specific detection: If studying IRF3 activation, consider phospho-specific antibodies that recognize specific phosphorylation sites.

• Species cross-reactivity: Human IRF3 shares 76% amino acid identity with mouse IRF3 and 83% with pig IRF3 over the region aa 206-427 . Consider this when planning cross-species studies.

• Application-specific performance: An antibody optimal for flow cytometry might not perform well in Western blotting or immunoprecipitation.

How can I validate IRF3 antibody specificity in my experimental system?

Rigorous validation of IRF3 antibody specificity should include:

• Genetic controls:

  • IRF3 knockout cells/tissues (CRISPR-Cas9 generated)

  • IRF3 siRNA or shRNA knockdown samples

  • Overexpression systems with tagged IRF3

• Peptide competition assays: Pre-incubate antibody with excess recombinant IRF3 protein (aa 206-427) before staining to verify specific binding .

• Orthogonal detection methods: Compare results using:

  • Multiple antibodies targeting different IRF3 epitopes

  • Alternative detection techniques (e.g., mass spectrometry)

  • mRNA expression correlation with protein detection

• Expected biological responses: Verify expected changes in:

  • Nuclear translocation following viral stimulation

  • Phosphorylation state changes after pathway activation

  • Interaction with known binding partners (e.g., STING pathway components)

• Cross-reactivity testing: Check for unexpected binding to related IRF family members, particularly IRF7 which shares structural similarities.

How should I interpret IRF3 staining patterns in different cell types and activation states?

Interpretation of IRF3 staining patterns requires understanding of:

• Subcellular localization patterns:

  • Resting cells: Predominantly cytoplasmic IRF3 distribution

  • Activated cells: Nuclear accumulation following phosphorylation

  • Partial activation: Mixed cytoplasmic and nuclear staining

• Cell type-specific considerations:

  • Daudi human Burkitt's lymphoma cells show detectable IRF3 levels suitable for flow cytometric analysis

  • Human plasmacytoid dendritic cells exhibit IRF3 regulation via the cGAS-STING pathway which affects TLR9-mediated IFN production

  • Primary cells vs. cell lines may show different baseline expression levels

• Activation-dependent phosphorylation:

  • Phospho-specific antibodies reveal activation state

  • Multiple phosphorylation sites (aa 395-407) may show different kinetics

  • Correlation between phosphorylation and nuclear translocation

• Expression level quantification:

  • Mean fluorescence intensity (MFI) can be used to quantify relative expression

  • Population heterogeneity may indicate different activation states

  • Comparison with isotype controls establishes specific staining thresholds

What are common sources of false positive or negative results when using IRF3 antibodies?

Researchers should be aware of potential artifacts and issues:

• False positives:

  • Insufficient blocking leading to non-specific binding

  • Spectral overlap in multicolor flow cytometry

  • Autofluorescence, particularly in tissue samples or certain cell types

  • Cross-reactivity with related IRF family proteins

  • Overfixation causing non-specific antibody trapping

• False negatives:

  • Epitope masking due to protein-protein interactions

  • Improper cell fixation/permeabilization preventing antibody access

  • Degradation of the antibody fluorophore due to light exposure

  • Buffer incompatibility affecting antibody binding

  • Low IRF3 expression levels below detection threshold

• Technical considerations:

  • Inappropriate antibody dilution

  • Suboptimal incubation conditions

  • Inadequate washing steps

  • Sample processing delays affecting protein preservation

How can I distinguish between different phosphorylation states of IRF3?

Distinguishing IRF3 phosphorylation states requires:

• Phospho-specific antibodies:

  • Select antibodies targeting specific phosphorylation sites within aa 395-407

  • Verify site-specificity with phospho-null and phospho-mimetic mutants

• Temporal analysis:

  • Perform time-course experiments following stimulation

  • Different phosphorylation sites may show distinct kinetics

• Correlative approaches:

  • Combine phospho-detection with subcellular localization analysis

  • Assess DNA binding activity in parallel with phosphorylation state

• Western blot analysis:

  • Phosphorylated IRF3 often shows mobility shift on SDS-PAGE

  • Use phosphatase treatment controls to confirm phosphorylation-dependent bands

• Mass spectrometry:

  • For definitive identification of specific phosphorylation sites

  • Can reveal novel or less-characterized modification patterns

How can IRF3 antibodies be used to study the cGAS-STING pathway in antiviral immunity?

The cGAS-STING pathway is a critical component of innate immune sensing that converges on IRF3 activation:

• Experimental design considerations:

  • Use IRF3 antibodies in combination with STING and cGAS antibodies for comprehensive pathway analysis

  • Examine IRF3 nuclear translocation as a readout for pathway activation

  • In human plasmacytoid dendritic cells, the cGAS-STING pathway has been shown to inhibit TLR9-mediated IFN production, requiring careful experimental design to distinguish these opposing effects

• Stimulation protocols:

  • Cytosolic DNA stimulation (e.g., dsDNA, cGAMP)

  • Viral infection models (DNA vs. RNA viruses)

  • Chemical agonists of STING (e.g., DMXAA in mouse cells)

• Readout systems:

  • Flow cytometry for phospho-IRF3 detection

  • Microscopy for IRF3 nuclear translocation

  • Reporter assays for IRF3-dependent transcription

• Validation approaches:

  • Genetic manipulation of pathway components (cGAS, STING, TBK1)

  • Pharmacological inhibitors of specific pathway nodes

  • siRNA-mediated knockdown of pathway components

What are the considerations for multiplexing IRF3 antibodies with other immune markers?

When designing multiplex experiments:

• Panel design principles:

  • Select compatible fluorophores with minimal spectral overlap

  • Human IRF3 Alexa Fluor® 488-conjugated Antibody can be combined with APC-conjugated antibodies such as Human/Mouse Frizzled-3 APC-conjugated Antibody with minimal compensation requirements

• Surface and intracellular marker combinations:

  • Perform surface staining before fixation and permeabilization

  • Verify that fixation/permeabilization protocols are compatible with all targeted epitopes

  • Consider sequential staining protocols for optimal results

• Pathway component co-detection:

  • Include upstream regulators (e.g., STING, TBK1)

  • Include downstream effectors (e.g., ISG products)

  • Consider including lineage markers for heterogeneous samples

• Controls for multiplex experiments:

  • Single-color controls for compensation

  • Fluorescence-minus-one (FMO) controls

  • Biological controls (stimulated vs. unstimulated)

• Data analysis considerations:

  • High-dimensional analysis techniques (tSNE, UMAP)

  • Boolean gating strategies for co-expression analysis

  • Correlation analysis between pathway components

How can I use IRF3 antibodies to investigate cross-talk between different innate immune signaling pathways?

IRF3 serves as an integration point for multiple innate immune pathways:

• Experimental strategies:

  • Sequential or combined pathway stimulation (e.g., TLR ligands plus STING agonists)

  • Inhibitor studies to block specific pathway branches

  • Time-course analysis to determine pathway kinetics and potential sequential activation

• Pathway interactions to consider:

  • cGAS-STING and TLR9 pathway antagonism in plasmacytoid dendritic cells

  • RIG-I/MDA5 and cGAS-STING cooperative effects

  • TLR3/4-TRIF and cytosolic sensing convergence

• Readout approaches:

  • Phospho-flow cytometry for IRF3 activation

  • Multiplex cytokine analysis for functional outcomes

  • Transcriptomics for pathway-specific gene signatures

• Validation methods:

  • Genetic deletion of specific pathway components

  • Reconstitution experiments in knockout systems

  • Inhibitor specificity controls

How do different classes of IRF3 antibodies compare for specific research applications?

Different IRF3 antibody formats offer distinct advantages:

When should I consider alternative approaches to antibody-based IRF3 detection?

Consider these complementary or alternative approaches:

• Genetic reporter systems:

  • IRF3-GFP fusion proteins for live-cell imaging

  • Luciferase reporters driven by IRF3-responsive promoters

  • Split-protein complementation assays for protein-protein interactions

• Mass spectrometry approaches:

  • For unbiased detection of IRF3 modifications

  • Interaction proteomics to identify binding partners

  • Absolute quantification of IRF3 levels

• RNA-based methods:

  • RT-qPCR for IRF3 transcript levels

  • RNA-seq for global IRF3-dependent transcriptional programs

  • Single-cell RNA analysis for heterogeneous responses

• CRISPR-based approaches:

  • IRF3 knockout/knockin validation

  • CRISPRa/CRISPRi for modulating IRF3 levels

  • CRISPR screens to identify IRF3 regulators

• In silico approaches:

  • Structural modeling of IRF3 interactions

  • Sequence conservation analysis across species

  • Network analysis of IRF3-dependent pathways

How can IRF3 antibodies be integrated with systems-level approaches to study innate immunity?

IRF3 antibodies can serve as critical tools in systems immunology:

• Single-cell analysis pipelines:

  • Combine IRF3 antibody staining with single-cell RNA-seq

  • CyTOF (mass cytometry) using metal-conjugated IRF3 antibodies

  • Spatial transcriptomics with IRF3 protein localization

• Multiomics integration:

  • Correlate IRF3 activation with global phosphoproteomics

  • Link IRF3 binding (ChIP-seq) with transcriptional outputs

  • Connect IRF3 activation states with metabolomic changes

• Mathematical modeling applications:

  • Use quantitative IRF3 data to parameterize computational models

  • Predict pathway dynamics based on IRF3 activation kinetics

  • Simulate perturbations to identify key regulatory nodes

• Translational research contexts:

  • Compare IRF3 activation patterns across patient cohorts

  • Correlate IRF3 activation with clinical outcomes

  • Assess therapeutic interventions targeting IRF3-dependent pathways