IRF7 Antibody, HRP conjugated

What is IRF7 and what is its significance in immune response research?

Interferon Regulatory Factor 7 (IRF7) is a transcription factor that functions as a master regulator of type I interferon responses during viral infections. IRF7 plays critical roles in both innate and adaptive immunity by regulating interferon production and mediating antigen presentation functionality. Research has identified IRF7 as essential for the production of type I interferons, particularly IFN-β, following viral detection . IRF7 deficiency has been linked to severe respiratory viral infections, including those caused by SARS-CoV-2, influenza virus, respiratory syncytial virus, and adenovirus, highlighting its importance in antiviral defense mechanisms . Beyond its role in infectious disease research, IRF7 has emerged as a significant factor in cancer research, particularly in prostate cancer where nuclear localization of IRF7 combined with PTEN-loss correlates with poorer patient outcomes .

How does an HRP-conjugated IRF7 antibody differ from unconjugated versions in experimental applications?

HRP (horseradish peroxidase)-conjugated IRF7 antibodies offer distinct advantages over unconjugated versions by eliminating the need for secondary antibody incubation in detection workflows. While unconjugated IRF7 antibodies (such as the 22392-1-AP) require a separate secondary antibody step , HRP-conjugated versions streamline experimental protocols by combining target recognition and enzymatic detection capabilities in a single reagent. This conjugation particularly benefits Western blot, immunohistochemistry (IHC), and ELISA applications by:

• Reducing protocol time and washing steps

• Minimizing cross-reactivity issues from secondary antibodies

• Enhancing signal consistency through fixed enzyme-to-antibody ratios

• Providing more direct quantification of target expression

When selecting between conjugated and unconjugated versions, researchers should consider their specific experimental design, the abundance of their target protein, and the detection sensitivity required.

What are the primary applications for IRF7 antibody in viral infection research?

IRF7 antibodies serve multiple crucial applications in viral infection research:

Research has demonstrated that IRF7 plays dual roles in corneal endothelial cells after HSV-1 infection by contributing to both type I interferon responses and mediating viral infection-induced MHC class I upregulation, which is essential for priming CD8 T cell immunity . This makes IRF7 antibodies valuable tools for studying both innate and adaptive immune responses to viral challenges.

What are the recommended dilution protocols for IRF7 antibody across different applications?

Optimal dilution of IRF7 antibody varies significantly by application methodology and specific experimental design. Based on established protocols for unconjugated antibodies (which provide reference points for HRP-conjugated versions):

For HRP-conjugated versions, initial dilutions should typically be higher (less concentrated) than unconjugated counterparts. Empirical determination through titration experiments remains essential for achieving optimal signal-to-noise ratios in each specific experimental system .

How can researchers validate IRF7 antibody specificity in their experimental systems?

Comprehensive validation of IRF7 antibody specificity requires multiple complementary approaches:

• Genetic validation: Use IRF7 knockdown/knockout systems as negative controls. In published research, IRF7 knockdown models showed impaired IFN-β production, confirming antibody specificity by demonstrating reduced signal .

• Stimulation studies: Treat cells with interferon or viral mimetics to upregulate IRF7 expression. Proper antibodies will show increased signal intensity correlating with activation stimulus duration/strength.

• Multi-application concordance: Verify consistent molecular weight detection across Western blot (approximately 55 kDa for IRF7), appropriate subcellular localization in immunofluorescence (cytoplasmic with nuclear translocation upon activation), and specific tissue staining patterns in IHC .

• Recombinant protein controls: Use purified recombinant IRF7 in Western blots and blocking experiments to confirm antibody recognition specificity.

• Cross-reactivity assessment: Test against tissue samples from IRF7-deficient models and closely related proteins (particularly other IRF family members) to evaluate potential cross-reactivity.

The gold standard validation includes rescue experiments where reintroduction of IRF7 expression in knockout systems restores antibody detection, as demonstrated in studies where "impaired IFN-β production by IRF7 ΔDBD was regained by IRF7 DNA transfection" .

What are the critical considerations when using IRF7 antibody to study nuclear translocation during viral infection?

Studying IRF7 nuclear translocation during viral infection requires particular methodological considerations:

• Temporal dynamics: IRF7 nuclear translocation occurs in a time-dependent manner following infection. Design time-course experiments capturing both early (0.5-2 hours) and later timepoints (6-24 hours) post-infection to document the complete translocation profile.

• Fixation methodology: Paraformaldehyde (4%) fixation preserves phosphorylation status and subcellular localization. Avoid methanol fixation which can disrupt phospho-epitopes critical for activated IRF7 detection.

• Fractionation controls: When performing nuclear/cytoplasmic fractionation for Western blot analysis, include markers for both compartments (e.g., HDAC1 for nuclear fraction, GAPDH for cytoplasmic fraction) to confirm separation quality.

• Quantification approach: For immunofluorescence studies, employ nuclear:cytoplasmic ratio quantification rather than simple positive/negative scoring. Research has shown that "a high ratio of nuclear:cytoplasmic IRF7 staining in cancers" correlates with prognosis in prostate cancer patients .

• Phosphorylation status: Consider using phospho-specific antibodies alongside total IRF7 antibodies to distinguish between inactive and activated forms.

• Confocal microscopy optimization: Use appropriate z-stack imaging to accurately capture nuclear versus perinuclear localization, which can be misinterpreted in standard epifluorescence microscopy.

How should researchers design experiments to investigate IRF7's role in antigen presentation?

Based on published research demonstrating IRF7's previously unrecognized role in MHC class I induction and antigen presentation , optimal experimental design should include:

• System preparation:

  • Generate IRF7-deficient cell models through CRISPR/Cas9 targeting of the DNA binding domain (similar to IRF7 ΔDBD models described in the literature)

  • Confirm functional disruption through type I IFN production assays

  • Prepare matched wild-type and IRF7-deficient cells for comparative analysis

• Viral infection model:

  • Select appropriate viral system (HSV-1 has been validated)

  • Determine optimal multiplicity of infection (MOI) and infection duration

  • Include UV-inactivated virus controls to distinguish between viral replication-dependent and independent effects

• MHC class I expression analysis:

  • Measure surface MHC class I levels by flow cytometry before and after infection

  • Perform Western blot analysis of total MHC class I protein

  • Quantify MHC class I mRNA through qPCR

• Functional antigen presentation assessment:

  • Develop co-culture systems with virus-specific memory CD8 T cells

  • Measure T cell activation markers and cytokine production

  • Quantify cytotoxic T lymphocyte (CTL) activity against infected cells

• Rescue experiments:

  • Reintroduce wild-type IRF7 to knockout models

  • Test domain-specific mutants to identify critical regions for MHC regulation

  • Validate restoration of both type I IFN production and MHC class I induction

This comprehensive approach would provide mechanistic insights into the dual role of IRF7 in both innate immunity (interferon production) and adaptive immunity (antigen presentation) .

What are the methodological considerations when using IRF7 antibodies in cancer research contexts?

When investigating IRF7 in cancer research, particularly prostate cancer where it has demonstrated prognostic value , researchers should consider:

• Expression pattern analysis:

  • Distinguish between nuclear and cytoplasmic IRF7 localization

  • Implement multiplex staining with other markers (e.g., PTEN) for prognostic assessment

  • Quantify nuclear:cytoplasmic IRF7 ratio through digital image analysis

• Survival signaling pathways:

  • Investigate IRF7's interaction with radiation response pathways

  • Monitor stem-like cell population changes after IRF7 manipulation

  • Assess type I interferon signaling network responses in IRF7-manipulated cancer models

• Therapeutic response prediction:

  • Correlate IRF7 expression patterns with treatment outcomes

  • Evaluate IRF7 as a biomarker for radiation therapy resistance

  • Investigate combination treatments targeting IRF7-dependent pathways

• Technical optimization:

  • For IHC applications, utilize TE buffer pH 9.0 for optimal antigen retrieval

  • Implement multiplex immunofluorescence to simultaneously visualize IRF7 and PTEN status

  • Develop quantitative scoring systems incorporating both intensity and localization

How can researchers troubleshoot weak or absent signals when using IRF7 antibody?

When encountering weak or absent IRF7 antibody signals, implement a systematic troubleshooting approach:

For research focusing on IRF7 in viral infection models, remember that IRF7 expression is dynamically regulated and peaks at specific timepoints post-infection. Capturing these temporal dynamics is essential for successful detection.

What strategies can enhance detection sensitivity when studying IRF7 in samples with low expression?

For samples with low IRF7 expression levels, several methodological enhancements can improve detection sensitivity:

• Signal amplification systems:

  • Employ tyramide signal amplification (TSA) for IHC/IF applications

  • Utilize high-sensitivity chemiluminescent substrates for Western blot

  • Consider biotin-streptavidin amplification systems for particularly challenging samples

• Sample enrichment:

  • Implement nuclear/cytoplasmic fractionation to concentrate IRF7

  • Utilize immunoprecipitation before Western blot analysis

  • Consider cell sorting to isolate relevant populations in heterogeneous samples

• Protocol optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Reduce washing stringency while maintaining specificity

  • Optimize blocking conditions to improve signal-to-noise ratio

• IRF7 induction:

  • When possible, stimulate samples with type I interferons or TLR agonists

  • Include positive control samples where IRF7 has been activated

• Detection system selection:

  • For Western blot: Select high-sensitivity femto-level detection substrates

  • For IHC: Implement polymer-based detection systems rather than traditional ABC methods

  • For IF: Utilize high-quantum-yield fluorophores and sensitive detection systems

These strategies are particularly important when studying IRF7 in non-lymphoid tissues or in conditions where its expression may be suppressed.

How can researchers optimize multiplex staining protocols including IRF7 antibody?

Optimizing multiplex protocols with IRF7 antibody requires careful consideration of several technical factors:

• Antibody compatibility assessment:

  • Verify primary antibodies originate from different host species

  • Test each antibody individually before combining

  • Confirm no cross-reactivity between detection systems

• Sequential staining approach:

  • Begin with the weakest signal (often IRF7 in non-stimulated conditions)

  • Implement heat or chemical stripping between rounds if using the same fluorophore class

  • Consider tyramide-based approaches for sequential same-species antibodies

• Panel design for IRF7 studies:

  • For viral studies: Include viral markers and IFN pathway components

  • For cancer research: Combine with PTEN staining as demonstrated in published research

  • For immune response characterization: Include cell-type markers with IRF7

• Imaging optimization:

  • Employ spectral unmixing for closely overlapping fluorophores

  • Collect single-stain controls for each fluorophore

  • Establish consistent exposure settings based on positive controls

• Quantification methodology:

  • Develop algorithms to quantify nuclear:cytoplasmic IRF7 ratios

  • Implement co-localization analysis for pathway interaction studies

  • Consider machine learning approaches for complex pattern recognition

Research has successfully employed multiplexed staining to simultaneously evaluate IRF7 and PTEN in cancer tissues, demonstrating that "a combined score comprising high nuclear IRF7 expression and low PTEN expression defined the worst prognosis cases" . This validates the feasibility and value of multiplex approaches with IRF7 antibodies.

How can IRF7 antibody be used to investigate genetic deficiencies related to viral susceptibility?

IRF7 antibodies are valuable tools for investigating genetic deficiencies associated with viral susceptibility:

• Clinical diagnostic applications:

  • Assess IRF7 protein expression in patients with severe or recurrent viral infections

  • Correlate IRF7 expression with genotype in patients carrying IRF7 variants

  • Monitor IRF7 activation in response to stimulation in patient-derived cells

• Variant characterization methodology:

  • Express identified IRF7 variants (e.g., R37H, T254A) in cellular models

  • Assess protein expression and stability by Western blot

  • Evaluate nuclear translocation capacity through immunofluorescence

  • Quantify transcriptional activity using reporter assays

• Functional immune phenotyping:

  • Analyze IFN-β production in patient cells after viral challenge

  • Assess IRF7 phosphorylation status using phospho-specific antibodies

  • Evaluate downstream interferon-stimulated gene induction

Research has identified that "autosomal recessive IRF7 deficiency was previously reported in three patients with single critical influenza or COVID-19 pneumonia episodes" and that these patients' cells "produced no detectable type I and III IFNs, except IFN-β" . These findings highlight the clinical relevance of IRF7 assessment in viral susceptibility evaluation.

What are the emerging applications of IRF7 antibody in cancer research and therapeutic development?

IRF7 antibodies are becoming increasingly important in cancer research:

• Prognostic biomarker development:

  • Evaluate nuclear IRF7 localization in tumor samples

  • Implement multiplexed assessment with PTEN and other markers

  • Correlate expression patterns with clinical outcomes

• Therapeutic resistance mechanisms:

  • Investigate IRF7's role in radiotherapy resistance

  • Examine connections between IRF7 and cancer stem cell populations

  • Assess IRF7-dependent type I interferon signaling in treatment response

• Therapeutic target validation:

  • Monitor IRF7 modulation after experimental treatments

  • Evaluate combination approaches targeting IRF7-dependent pathways

  • Develop screening platforms for compounds affecting IRF7 activity

• Precision medicine applications:

  • Stratify patients based on IRF7/PTEN expression profiles

  • Tailor treatment approaches based on IRF7 status

  • Monitor treatment efficacy through changes in IRF7 activation

Research has demonstrated that IRF7 "is a key mediator of this stress-responsive signature and is required for the occurrence of stem-like populations in response to low-dose fractionated radiation in vitro and in vivo" and that "IRF7 can support both the expression of a network of stress responsive Type1 interferon genes and also the survival of cells treated with radiation" . These findings position IRF7 as a potential therapeutic target and biomarker in cancer treatment.

How can researchers integrate IRF7 antibody-based detection with transcriptomic and proteomic approaches?

Integrating IRF7 antibody-based detection with multi-omics approaches enables comprehensive pathway analysis:

• Correlative experimental design:

  • Design studies that collect matched samples for antibody-based detection, transcriptomics, and proteomics

  • Implement time-course analyses to capture dynamic regulation

  • Include appropriate cellular fractionation for nuclear versus cytoplasmic signaling

• Methodological integration:

  • Validate transcriptomic findings at protein level using IRF7 antibodies

  • Correlate post-translational modifications identified in proteomics with IRF7 activation status

  • Use antibody-based sorting/enrichment before omics analysis to focus on IRF7-expressing populations

• Data integration approaches:

  • Implement pathway analysis incorporating IRF7-dependent genes

  • Develop computational models integrating transcriptomic, proteomic, and functional data

  • Utilize network analysis to identify key nodes in IRF7-mediated responses

Research has successfully implemented such integrated approaches, demonstrating that "our 'comprehensive' network highlights biological processes including viral immune response, proliferation (nucleosome assembly) and keratins" and that "the irradiated control (IRF7 wild type) network contained 13 genes of the 14 genes- 'IRF7-Sig'" . These integrated approaches provide mechanistic insights beyond what can be achieved with antibody-based detection alone.

What are the critical parameters for optimizing Western blot protocols with IRF7 antibody?

Optimizing Western blot protocols for IRF7 detection requires attention to several critical parameters:

• Sample preparation:

  • Include phosphatase inhibitors to preserve activation-dependent phosphorylation

  • Consider nuclear/cytoplasmic fractionation for analyzing translocation

  • Maintain cold chain throughout lysis to prevent degradation

• Gel percentage selection:

  • Use 8-10% gels for optimal resolution around IRF7's 55-56 kDa molecular weight

  • Consider gradient gels when analyzing both IRF7 and its interaction partners

• Transfer optimization:

  • Implement wet transfer for larger proteins like IRF7

  • Optimize transfer time and voltage for complete transfer

  • Verify transfer efficiency with reversible stains

• Blocking and antibody conditions:

  • Test both BSA and milk-based blocking solutions

  • Implement extended primary antibody incubation (overnight at 4°C)

  • Determine optimal dilution through titration (1:2000-1:16000 range)

• Detection considerations:

  • Select detection reagents appropriate for expression level

  • Include positive controls (HEK-293 cells, mouse/rat kidney tissue)

  • Implement stripping and reprobing for multiple targets

These optimizations are particularly important when analyzing IRF7 in experimental systems where its expression may be dynamically regulated or present at low levels.

What are the key methodological considerations for studying IRF7 in viral infection models?

When employing IRF7 antibodies in viral infection research models:

• Temporal dynamics:

  • Design time-course experiments capturing IRF7 induction and activation

  • Include early timepoints (2-6 hours) to capture initial responses

  • Extend analysis to later timepoints (24-72 hours) for adaptive response

• Cell type considerations:

  • Recognize cell-type specific differences in IRF7 basal expression

  • Include relevant controls (e.g., IRF7-deficient cells) for each cell type

  • Consider primary cells versus cell lines for physiological relevance

• Viral strain selection:

  • Choose appropriate viral systems (HSV-1, influenza, SARS-CoV-2)

  • Include UV-inactivated controls to distinguish replication-dependent effects

  • Consider MOI optimization for each experimental endpoint

• Functional correlates:

  • Pair IRF7 detection with interferon production measurement

  • Assess antigen presentation capacity as demonstrated in published research

  • Evaluate downstream interferon-stimulated gene expression

• Therapeutic context:

  • Design intervention studies (timing, dose) based on IRF7 activation kinetics

  • Consider combinatorial approaches targeting multiple pathway components

  • Include clinically relevant endpoints beyond molecular markers