IRF5 Antibody

What is IRF5 and why is it important in immunological research?

IRF5 is a potent pro-inflammatory transcription factor that orchestrates transcription of inflammatory mediators and polarizes macrophages to a Th1/17 inducing phenotype. It plays a crucial role in TLR signaling pathways and forms heterodimers with IRF3 for interferon gene transcription . IRF5 knockout mice studies demonstrate its critical role in apoptosis induction .

IRF5 has gained significant research interest due to its genetic association with multiple autoimmune diseases. Single nucleotide polymorphisms in the IRF5 gene are strongly linked to systemic lupus erythematosus (SLE), rheumatoid arthritis, and inflammatory bowel disease . IRF5 has been identified as one of the most significant expression quantitative trait loci (eQTL) in human myeloid cells stimulated with LPS, highlighting its importance as a key regulator of TLR-induced inflammatory responses .

Recent studies have expanded our understanding of IRF5's role beyond autoimmunity to neuropathic pain, obesity, myocardial infarction, allograft rejection, atherosclerosis, and metabolic dysfunction, making it a critical target for various fields of biomedical research .

How do I select the appropriate IRF5 antibody for my experiments?

Selecting the appropriate IRF5 antibody requires careful consideration of both application specificity and validation status. Research has shown that many commercial antibodies against IRF5 lack specificity or perform inconsistently across applications .

Based on published validation studies, these antibodies have demonstrated reliable performance in specific applications:

Always verify that your selected antibody has been validated using appropriate positive controls (THP1 monocytes, Ramos B cells) and negative controls (Jurkat T cells, IRF5 knockout cells) . Consider whether the antibody has been validated for your specific application and whether it recognizes your species of interest, as cross-species reactivity cannot be assumed.

What are the key considerations for validating IRF5 antibody specificity?

Validating IRF5 antibody specificity is crucial for obtaining reliable results. Research has shown that many commercially available antibodies detect bands in negative controls or fail to detect differences after IRF5 knockdown .

Essential validation steps include:

• Use of proper controls: Include both positive (THP1, Ramos B cells) and negative (Jurkat T cells, IRF5 knockout cells) controls in each experiment .

• siRNA knockdown verification: This approach confirms that observed bands are specifically IRF5. In validation studies, antibodies ab33478 and ab2932 were unable to detect differences in IRF5 expression after knockdown, while cs3257 reliably showed decreased IRF5 expression .

• Cross-application testing: An antibody might perform well in one application but poorly in others. For example, ab124792 proved excellent for immunoprecipitation despite manufacturer claims to the contrary .

• Molecular weight verification: Human IRF5 exists in multiple isoforms giving bands between 48-62 kDa. Bands outside this range may represent non-specific binding or post-translational modifications .

• Tissue-specific validation: For tissue sections, co-staining with lineage markers (e.g., CD3 for T cells) helps confirm specificity of IRF5 staining patterns. Studies have shown that antibodies like ab124792 correctly showed IRF5 positivity in CD3-negative cells in human spleen .

How can I optimize IRF5 detection by flow cytometry?

Flow cytometric detection of IRF5 requires careful optimization of fixation, permeabilization, and antibody selection. Research has identified several antibodies that perform well in this application, with ab124792 showing the largest difference between positive and negative populations based on mean fluorescence intensity (MFI) .

For optimal intracellular staining of IRF5:

• Fixation protocol: Use a dedicated flow cytometry fixation buffer (such as Flow Cytometry Fixation Buffer) followed by permeabilization with an appropriate buffer (like Flow Cytometry Permeabilization/Wash Buffer I) .

• Antibody selection and titration: Validated antibodies include cs3257, ab124792, cs13496, and SAB1403991 . Titrate your selected antibody to determine optimal concentration - too much antibody increases background, while too little reduces sensitivity.

• Controls: Include an isotype control antibody at the same concentration as your IRF5 antibody. For biological controls, use IRF5-negative cell lines like Jurkat T cells alongside IRF5-expressing lines like THP1 or Ramos B cells .

• Panel design considerations: When using multiple colors, include single-stained controls for compensation. IRF5 is primarily cytoplasmic in resting cells but can translocate to the nucleus upon activation, so consider nuclear markers in your panel if studying activation states.

• Enhanced detection: Pre-conjugated forms of ab124792 (such as ab193245 or ab192983) have demonstrated improved specificity for intracellular IRF5 detection .

What are the best practices for immunoprecipitation of IRF5?

Immunoprecipitation (IP) of IRF5 presents unique challenges, as many antibodies recommended for IP either fail to efficiently pull down IRF5 or exhibit non-specific binding .

Through extensive testing, these key practices have emerged:

• Antibody selection: Surprisingly, ab124792 has demonstrated excellent IP efficiency and specificity for IRF5 despite manufacturer claims of unsuitability for this application . Clone 2E3-1A11 (WH0003663M1) also showed good efficiency, while cs3257, which performs well in Western blot, showed poor IP efficiency .

• Optimized protocol:

  • Use 4 μg of ab124792 antibody per standard IP reaction (500-1000 μg total protein)

  • Include stringent wash steps to reduce non-specific binding

  • Confirm specificity by performing parallel IPs with IRF5-negative cells (e.g., Jurkat T cells)

  • Validate results by immunoblotting IP products with a different IRF5 antibody

• Validation approach: A key experiment showed that when identical conditions were used, ab124792 successfully immunoprecipitated IRF5 from Ramos B cells (IRF5-positive) but showed no binding in Jurkat T cells (IRF5-negative), confirming specificity .

• Co-immunoprecipitation considerations: If studying IRF5 interaction partners, gentler wash conditions may preserve protein-protein interactions at the cost of potentially increased background.

This systematic optimization is essential as IP efficiency can vary dramatically between antibodies even when they perform similarly in other applications.

How should I approach IRF5 detection in tissue sections?

Immunohistochemical (IHC) detection of IRF5 in tissues requires careful consideration of antibody selection, staining protocols, and proper controls:

• Antibody validation: Several antibodies have been validated for IRF5 detection in human tissues. Ab124792 and ab181553 showed similar staining patterns in human spleen, with higher IRF5 expression in red pulp compared to white pulp . HPA046700 showed a distinct pattern with higher expression in white pulp .

• Proper controls: Co-staining with lineage markers provides critical validation. For example, IRF5 expression in spleen was confirmed using CD3 co-staining, which demonstrated that IRF5 staining (with ab124792) occurred in CD3-negative cells, consistent with IRF5's known expression pattern .

• Tissue preparation considerations:

  • Formalin-fixed paraffin-embedded tissues require appropriate antigen retrieval

  • Fresh frozen tissues often provide better epitope preservation

  • Background reduction through blocking of endogenous peroxidase activity is essential

• Species specificity: Ensure your antibody has been validated in your species of interest. Many antibodies show different specificities between human and murine tissues .

• Pathological assessment: In disease states like SLE, IRF5 expression patterns may change. Studies have shown altered IRF5 activation in both active disease and remission phases . When examining pathological tissues, include both disease and normal control tissues for comparison.

How do I interpret multiple bands in Western blots with IRF5 antibodies?

Multiple bands in IRF5 Western blots require careful analysis to distinguish specific from non-specific signals:

• Expected IRF5 isoforms: Human IRF5 has multiple splice variants (V1-V9) ranging from approximately 48-62 kDa, with predominant variants V1, V2, V3, and V5 . Different isoforms may be expressed in different cell types or disease states.

• Validation through knockdown: siRNA knockdown experiments are essential to confirm which bands represent IRF5. Research has shown that antibodies like cs3257 detect bands that diminish after IRF5 knockdown, while other antibodies (e.g., ab33478 and ab2932) detect bands that remain unchanged, indicating non-specific binding .

• Post-translational modifications: Phosphorylated IRF5 and other modified forms may appear as higher molecular weight bands. These modifications increase following cellular activation.

• Non-specific binding assessment: Compare observed bands between positive controls (THP1, Ramos B cells) and negative controls (Jurkat T cells, IRF5 knockout cells). Some antibodies (e.g., SAB1403991) detect both specific IRF5 bands and non-specific proteins .

• Multiple antibody approach: Using multiple antibodies targeting different IRF5 epitopes can help confirm band identity. Consistent bands across different antibodies are more likely to represent true IRF5 signal.

What controls are essential when studying IRF5 in disease models?

• Genetic controls:

  • IRF5 knockout mice provide the gold standard negative control for specificity

  • Conditional IRF5 knockout models can dissect cell-specific contributions to pathology

  • For human studies, cells with IRF5 knockdown via siRNA serve as important controls

• Disease model controls:

  • Include both disease-induced and non-induced animals in the same experiment

  • For SLE models, compare pristane-injected IRF5-deficient and wild-type mice

  • Time-course experiments can distinguish between acute and chronic phases of disease

• Treatment intervention controls:

  • When testing IRF5 inhibitors, include both pre-disease and post-disease onset intervention groups

  • Compare IRF5 inhibition to established therapies (e.g., type I interferon blockade)

  • Include vehicle-treated controls with matched timing and administration routes

• Human sample controls:

  • Compare active disease, remission, and healthy control samples

  • Match controls for age, sex, and treatment status when possible

  • For tissue analyses, include both affected and unaffected tissues from the same patient

• Antibody validation in disease context:

  • Revalidate antibody specificity in disease tissues, as inflammation can alter staining patterns

  • Include isotype controls at matched concentrations

  • Consider the impact of treatments on IRF5 detection (some therapies may modify epitopes)

Research has shown that IRF5 knockout mice fail to develop antinuclear autoantibodies and renal immune complex deposits in SLE models, highlighting the value of genetic controls .

What parameters should I optimize when investigating IRF5 activation?

IRF5 activation involves multiple molecular events that require specific optimization parameters:

• Stimulation conditions:

  • TLR ligands (particularly TLR7/8 and TLR9) are potent IRF5 activators

  • Immune complexes containing nucleic acids activate IRF5 in SLE models

  • Time-course experiments are essential as IRF5 activation is dynamic (typically examine 15min-24h post-stimulation)

  • Dose-response studies help identify optimal stimulation conditions

• Activation readouts:

  • Nuclear translocation (cytoplasmic-to-nuclear shift)

  • Phosphorylation state changes

  • Binding to target gene promoters

  • Induction of IRF5-dependent genes (type I interferons, inflammatory cytokines)

• Technical considerations:

  • For nuclear translocation, optimize nuclear/cytoplasmic fractionation protocols

  • For phosphorylation, include phosphatase inhibitors in all buffers

  • For chromatin studies, optimize crosslinking and sonication conditions

  • For gene expression, select appropriate housekeeping genes as normalizers

• Controls for activation studies:

  • Include IRF5-deficient cells to confirm signal specificity

  • Use inhibitors of upstream pathways (e.g., TLR pathway inhibitors)

  • Compare IRF5 wildtype to variant forms associated with autoimmunity

  • Include unstimulated controls at each time point

• Disease-relevant conditions:

  • For SLE studies, patient-derived immune complexes provide physiologically relevant stimuli

  • Compare cells from patients with different IRF5 risk haplotypes

  • Assess the impact of other disease-relevant factors (e.g., type I interferons, cytokines)

Research has shown that SLE patients exhibit aberrant IRF5 activation not only during active disease but also in remission phases, suggesting constitutive dysregulation .

How does IRF5 contribute to SLE pathogenesis?

IRF5 plays a central role in SLE pathogenesis through multiple mechanisms:

• Genetic predisposition: IRF5 polymorphisms are strongly associated with SLE susceptibility in humans . These variants affect both expression level and isoform ratios, creating a genetic foundation for disease risk.

• B cell dysfunction: IRF5 is required for class switch recombination to IgG2a in mice (analogous to human IgG1/IgG3), which are the predominant pathogenic autoantibody isotypes in SLE . IRF5-deficient mice fail to develop antinuclear autoantibodies and glomerular immune complex deposits when challenged with pristane . B cell-intrinsic IRF5 function is critical for autoantibody production in mouse models .

• Type I interferon production: IRF5 drives transcription of type I interferons, particularly IFN-α . SLE patients show elevated interferon-stimulated gene expression, known as the "interferon signature." This creates a feed-forward loop where immune complexes containing self-nucleic acids activate IRF5 through TLR7/9, leading to more autoantibody production .

• Inflammatory cytokine regulation: IRF5 orchestrates production of pro-inflammatory cytokines and polarizes macrophages toward an inflammatory phenotype . These inflammatory mediators contribute to tissue damage and perpetuate autoimmunity.

• Therapeutic implications: Studies show that IRF5 inhibition may be superior to blocking type I interferon signaling in SLE models, potentially due to IRF5's additional roles in oxidative phosphorylation . Importantly, conditional IRF5 deletion and small-molecule IRF5 inhibitors can suppress disease progression even after disease onset .

This multifaceted involvement makes IRF5 a promising therapeutic target for SLE and potentially other autoimmune diseases.

What experimental methods are used to study IRF5 polymorphisms in disease?

Studying IRF5 polymorphisms in disease contexts requires specialized experimental approaches:

• Genotyping technologies:

  • SNP genotyping arrays to identify IRF5 risk variants

  • Targeted sequencing of the IRF5 locus

  • Expression quantitative trait loci (eQTL) analysis to link variants to expression patterns

  • Haplotype determination to assess combined effects of multiple variants

• Functional genomics approaches:

  • Allele-specific expression analysis

  • CRISPR-Cas9 editing to create or repair risk variants

  • Reporter assays with variant promoter/enhancer elements

  • RNA-seq to assess transcriptome-wide effects of variants

• Proteomic analysis:

  • Quantitative Western blotting to measure isoform expression differences

  • Mass spectrometry to characterize IRF5 isoforms and modifications

  • Protein-protein interaction studies to identify variant-specific binding partners

  • Structural analysis of variant IRF5 proteins

• Cellular phenotyping:

  • Flow cytometry with validated antibodies to assess IRF5 expression in different cell types

  • Ex vivo stimulation assays to measure variant-specific activation thresholds

  • Cytokine profiling to characterize functional consequences of variants

  • Cell type-specific responses to TLR ligands or immune complexes

• Clinical correlations:

  • Association of IRF5 variants with specific disease manifestations

  • Longitudinal studies correlating variants with disease progression

  • Treatment response prediction based on IRF5 genotype

  • Biomarker development for IRF5 activity in patient samples

These approaches have revealed that IRF5 risk variants alter both the quantity and quality of IRF5 protein, affecting its function in multiple immune cell types and contributing to disease pathogenesis .

How might IRF5 inhibition be developed as a therapeutic strategy?

IRF5 inhibition represents a promising therapeutic avenue for autoimmune diseases, particularly SLE:

• Advantages over current approaches:

  • Research demonstrates that partial IRF5 inhibition can be superior to complete blockade of type I interferon signaling in SLE models

  • IRF5 inhibition suppresses disease progression even when administered after disease onset

  • IRF5 inhibition is effective for maintenance of remission in mouse models

  • Targeting IRF5 addresses multiple pathogenic mechanisms simultaneously (autoantibody production, type I interferon, inflammatory cytokines)

• Inhibition strategies:

  • Small-molecule inhibitors that target IRF5 directly

  • Peptide-based inhibitors of IRF5 dimerization or DNA binding

  • Oligonucleotide-based approaches to reduce IRF5 expression

  • Cell-specific delivery systems to target relevant immune cell populations

• Development considerations:

  • Partial versus complete inhibition (complete loss may impair antimicrobial defense)

  • Cell type-specific effects of inhibition

  • Timing of intervention (prevention, active disease, maintenance therapy)

  • Biomarkers to monitor IRF5 activity and predict/assess response

• Combination approaches:

  • IRF5 inhibition plus B cell-targeted therapies

  • IRF5 inhibition with cytokine blockade

  • Sequential therapy strategies (induction with conventional agents, maintenance with IRF5 inhibition)

• Translational research needs:

  • Development of human IRF5 activity biomarkers

  • IRF5 genotype influence on treatment response

  • Safety evaluation regarding infection risk and tumor surveillance

  • Patient stratification strategies for clinical trials

Research has shown that both genetic (conditional knockout) and pharmacological (small-molecule inhibitor) approaches to IRF5 inhibition are effective in murine SLE models, providing proof-of-concept for therapeutic development .

Why might my IRF5 Western blot show inconsistent results?

Inconsistent Western blot results with IRF5 antibodies can stem from several factors:

• Antibody specificity issues: Many commercial IRF5 antibodies lack specificity or detect non-specific bands. Research has identified that antibodies like ab33478 and ab2932 fail to distinguish between IRF5-expressing and non-expressing cells .

• Sample preparation variables:

  • Incomplete protein denaturation affects epitope exposure

  • Different lysis buffers may extract IRF5 with varying efficiency

  • Phosphatase inhibitor omission can alter the migration pattern

  • Protein degradation during preparation creates misleading fragments

• Technical execution factors:

  • Transfer efficiency variations, especially for higher molecular weight proteins

  • Inconsistent blocking conditions leading to background differences

  • Development timing affecting band intensity

  • Loading control quality and normalization approach

• IRF5 biology complexities:

  • Multiple isoform expression varies between cell types and conditions

  • Post-translational modifications change upon cell activation

  • IRF5 expression level changes with cellular state

  • Cell type-specific expression patterns

• Validated troubleshooting approaches:

  • Use antibodies with confirmed specificity (cs3257, ab124792, ab181553)

  • Include positive controls (THP1, Ramos B cells) and negative controls (Jurkat T cells)

  • Run IRF5 knockdown samples alongside experimental samples

  • Consider native versus reducing conditions for epitope preservation

Researchers have found that using validated antibodies and rigorously controlling experimental conditions significantly improves reproducibility in IRF5 Western blot experiments.

How can I improve the specificity of IRF5 immunostaining?

Improving IRF5 immunostaining specificity requires a systematic approach:

• Antibody selection: Research has identified ab124792 and ab181553 as highly specific for IRF5 immunostaining in human tissues . These antibodies showed consistent staining patterns in human spleen, with higher IRF5 expression in red pulp compared to white pulp.

• Protocol optimization:

  • Titrate primary antibody concentration to minimize background

  • Extend blocking steps (1-2 hours at room temperature or overnight at 4°C)

  • Increase wash duration and number after primary and secondary antibody steps

  • Optimize antigen retrieval methods for fixed tissues

  • Reduce secondary antibody concentration if background persists

• Validation approaches:

  • Perform co-staining with lineage markers (e.g., CD3 for T cells) to verify cell type-specific patterns

  • Include isotype control antibodies at matched concentration

  • Use IRF5-deficient tissues when available

  • Pre-absorb antibody with recombinant IRF5 protein when possible

  • Compare staining patterns across multiple antibodies targeting different epitopes

• Tissue-specific considerations:

  • Optimize fixation protocols for your specific tissue type

  • Address tissue-specific autofluorescence (using methods like Sudan Black treatment)

  • Consider tissue-specific blocking reagents (e.g., avidin/biotin blocking for high biotin tissues)

  • Validate in multiple donors/samples to account for individual variation

• Advanced techniques:

  • Consider signal amplification methods for low-abundance detection

  • Use spectral imaging to distinguish true signal from autofluorescence

  • Employ multiplexed approaches to assess co-localization with known markers

  • Consider proximity ligation assays for improved specificity

Verification with co-staining approaches has proven valuable, as demonstrated by studies showing that ab124792 stained positive for IRF5 in cells lacking CD3 co-stain in human spleen, confirming appropriate cell type specificity .

What strategies can address the challenge of detecting IRF5 in primary cells?

Detecting IRF5 in primary cells presents unique challenges that require specialized approaches:

• Cell type-specific considerations:

  • IRF5 expression varies substantially between immune cell populations

  • Highest expression in plasmacytoid dendritic cells, B cells, and monocytes

  • Lower expression in conventional dendritic cells and macrophages

  • Minimal expression in resting T cells and many non-immune cells

  • Expression may change dramatically upon activation or in disease states

• Sample preparation optimization:

  • Rapid processing to preserve protein integrity

  • Optimized fixation/permeabilization for primary cells

  • Enrichment of target cell populations through sorting or magnetic separation

  • Appropriate activation protocols to enhance detection if studying inducible expression

• Detection method selection:

  • Flow cytometry with validated antibodies (ab124792, cs3257) for quantitative single-cell analysis

  • Immunofluorescence microscopy for localization studies

  • Highly sensitive Western blot protocols for limited cell numbers

  • qPCR for mRNA expression correlation with protein analysis

• Protocol adaptations:

  • Increased antibody incubation time (overnight at 4°C)

  • Signal amplification systems for low-abundance detection

  • Cell surface marker co-staining to identify specific populations

  • Nuclear counterstaining to assess localization state

• Validation approaches:

  • Compare detection in purified cell populations versus mixed samples

  • Correlate protein detection with mRNA expression

  • Use stimulation conditions known to alter IRF5 expression/localization

  • Include matched control subjects alongside patients/experimental groups

These approaches have been successfully applied in research examining IRF5 in primary cells from SLE patients, revealing that both active disease and remission phases show aberrant IRF5 activation and interferon-stimulated gene expression .