IRF5 Antibody

What is IRF5 and why is it important in research?

IRF5 is a transcription factor that plays a critical role in innate immunity by activating expression of type I interferons (IFN-alpha and IFN-beta) and inflammatory cytokines downstream of endolysosomal toll-like receptors TLR7, TLR8, and TLR9 . IRF5 regulates gene transcription by binding to interferon-stimulated response elements (ISREs) in promoters . Beyond innate immunity, IRF5 has been implicated in autoimmunity (particularly SLE/lupus), cancer, obesity, pain, cardiovascular disease, and metabolism . Its diverse functions make it a significant target for immunological and disease-focused research.

How does IRF5 function in B cells specifically?

IRF5 plays a B cell-intrinsic role in antibody production and class switching. Research demonstrates that IRF5 is required for the secretion of IgG2a antibodies during immune responses to T-cell-dependent antigens . In human B cells, IRF5 is essential in the early stages of B cell activation and proliferation in response to TLR9/BCR-induced antibody-secreting cell (ASC) differentiation . IRF5 knockdown in human B cells results in significant increases in IgD+ B cells, reduced activation, decreased clonal expansion, impaired plasmablast differentiation, and reduced IgG1/3 secretion . IRF5 also regulates expression of activation-induced cytidine deaminase (AID), which is crucial for class-switch recombination .

What are the known variants or isoforms of IRF5?

While the search results don't explicitly detail all IRF5 isoforms, it's important to note that IRF5 exists in multiple isoforms that may have different functions or expression patterns. When selecting antibodies, researchers should consider whether the antibody recognizes all relevant isoforms or is specific to particular variants. The main IRF5 protein is reported to be approximately 56 kilodaltons in mass .

How should I validate the specificity of IRF5 antibodies?

Antibody validation is critical as many commercial IRF5 antibodies lack specificity. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known IRF5 expression levels (e.g., Ramos B cells as positive controls)

• Genetic controls: Utilize IRF5 knockout mice or tissues as negative controls

• siRNA knockdown: Confirm specificity by knocking down IRF5 in cells that express it and demonstrating reduced detection

• Molecular weight verification: Confirm bands appear at the expected molecular weight (approximately 56 kDa)

• Cross-application validation: Test antibodies in multiple applications (WB, flow cytometry, IF) to determine application-specific performance

Research has shown that many commercial antibodies detect signals in IRF5 knockout samples or fail to detect differences after IRF5 knockdown, indicating poor specificity .

Which IRF5 antibodies have been experimentally validated for specific applications?

Based on the search results, the following antibodies have demonstrated specific detection of IRF5 in particular applications:

Interestingly, some antibodies like cs3257 that work well for western blot were unable to efficiently immunoprecipitate IRF5, highlighting the importance of application-specific validation .

What are the most common pitfalls in IRF5 antibody-based experiments?

Common pitfalls include:

• Lack of proper controls: Failing to include positive and negative controls leads to misinterpretation of non-specific signals

• Cross-reactivity: Many antibodies detect non-IRF5 proteins of similar molecular weight

• Application mismatch: Using antibodies for applications they haven't been validated for

• Inappropriate interpretation: Attributing multiple bands to isoforms without validation

• Inadequate documentation: Not including molecular weight standards in published results

According to validation studies, the majority of commercial IRF5 antibodies tested were unable to specifically recognize human or mouse IRF5, which can significantly compromise research findings .

How can IRF5 antibodies be used to study autoimmune disease mechanisms?

IRF5 antibodies can be valuable tools for studying autoimmune diseases, particularly SLE, by:

• Assessing IRF5 expression levels: Comparing IRF5 expression between healthy controls and patients with autoimmune diseases

• Examining IRF5 activation status: Using phospho-specific antibodies to determine IRF5 activation

• Analyzing IRF5's role in B cell differentiation: Given IRF5's importance in B cell activation and antibody production, antibodies can be used to track IRF5 during B cell differentiation stages in autoimmune contexts

• Correlating with autoantibody production: Using IRF5 antibodies alongside assays for autoantibodies (like ANAs) to establish relationships

Research has shown that IRF5 influences autoantibody production in SLE, particularly IgG2a and IgG2b ANAs, which are critical for disease development . IRF5 knockout mice show reduced anti-dsDNA and anti-sm autoantibodies in SLE models .

How does IRF5 signaling differ between cell types and how can antibodies help characterize these differences?

IRF5 functions differently across cell types, and antibodies can help characterize these differences through:

• Flow cytometry: Measuring IRF5 expression levels in different immune cell populations

• Immunofluorescence: Examining subcellular localization of IRF5 in different cell types

• Chromatin immunoprecipitation (ChIP): Identifying cell-type specific IRF5 binding sites

• Co-immunoprecipitation: Identifying cell-type specific IRF5 binding partners

While IRF5's role is well-characterized in monocytes and dendritic cells, its function in B cells remains less defined . Human B cell studies show IRF5 regulates different target genes compared to murine studies, including IRF4, ERK1, and MYC .

What are the key methodological considerations for studying IRF5 phosphorylation and activation?

When studying IRF5 activation:

• Phospho-specific antibodies: Select antibodies that recognize specific phosphorylation sites associated with IRF5 activation

• Nuclear-cytoplasmic fractionation: Combine with western blotting to track IRF5 nuclear translocation upon activation

• Timing considerations: IRF5 activation occurs rapidly after TLR stimulation, so establish appropriate time courses

• Pathway inhibitors: Use in conjunction with antibodies to confirm specificity of activation signals

• Cross-validation: Confirm activation using multiple methods (phosphorylation, nuclear translocation, target gene expression)

While the search results don't specifically address IRF5 phosphorylation methodologies, IRF5's role downstream of TLR7, TLR8, and TLR9 signaling implies that stimulation with appropriate ligands would induce IRF5 phosphorylation and activation .

How can I resolve inconsistent results when using IRF5 antibodies across different applications?

Inconsistent results often stem from application-specific performance issues. To resolve these:

• Application-specific validation: Test each antibody in your specific application with proper controls

• Buffer optimization: Modify lysis, blocking, or wash buffers to improve specificity

• Signal enhancement techniques: For weak signals, consider using amplification systems without compromising specificity

• Cross-antibody verification: Use multiple antibodies targeting different epitopes to confirm results

• Epitope accessibility: Consider whether sample preparation might affect epitope availability

Research has demonstrated that antibodies perform differently across applications; for example, cs3257 worked well for western blot but poorly for immunoprecipitation, while clone 2E3-1A11 excelled at immunoprecipitation .

What is the best approach for detecting IRF5 in tissue samples?

For optimal IRF5 detection in tissues:

• Validated antibodies: Use only antibodies specifically validated for immunohistochemistry/immunofluorescence

• Fixation optimization: Test different fixation methods to preserve epitope recognition

• Antigen retrieval: Optimize antigen retrieval protocols for IRF5 detection

• Signal amplification: Consider tyramide signal amplification for low abundance detection

• Multiplex staining: Combine IRF5 staining with cell type markers to identify specific expressing populations

• Controls: Include IRF5 knockout tissues or IRF5-negative tissues as negative controls

While specific protocols aren't detailed in the search results, they emphasize the importance of validation with proper controls for tissue-based applications .

How can RNA interference techniques be used to validate IRF5 antibody specificity?

RNA interference provides powerful validation for antibody specificity:

• siRNA targeting: Design siRNAs targeting multiple regions of IRF5 mRNA

• Transfection optimization: Optimize transfection conditions for your cell type

• Quantification: Confirm knockdown efficiency by qRT-PCR (typically aim for >70% reduction)

• Timing: Allow sufficient time for protein turnover (typically 48-72 hours)

• Multiple siRNAs: Use multiple siRNA sequences to control for off-target effects

• Antibody testing: Compare antibody signals between control and knockdown samples

Researchers have successfully used siRNA to knockdown IRF5 in Ramos B cells, demonstrating that certain antibodies (cs3257, cs13496) could detect the resulting reduction in IRF5 expression while others (ab33478, ab2932) could not .

How can IRF5 antibodies be used in single-cell analysis techniques?

IRF5 antibodies can be integrated into single-cell analyses through:

• Mass cytometry (CyTOF): Using metal-conjugated IRF5 antibodies for high-parameter single-cell profiling

• Single-cell western blot: Detecting IRF5 expression in individual cells

• Imaging mass cytometry: Visualizing IRF5 expression in tissue contexts at single-cell resolution

• Flow cytometry: Using fluorophore-conjugated IRF5 antibodies in multi-parameter panels

• CITE-seq: Combining transcriptomics with antibody-based protein detection

These approaches could help identify IRF5 expression heterogeneity within seemingly homogeneous cell populations and correlate IRF5 expression with other markers at the single-cell level.

What are the considerations for using IRF5 antibodies in systems biology approaches?

When incorporating IRF5 antibodies into systems biology studies:

• Multi-omics integration: Combine antibody-based IRF5 detection with transcriptomics, proteomics, and epigenomics

• Network analysis: Use co-immunoprecipitation with IRF5 antibodies to map protein-protein interaction networks

• ChIP-seq: Map genome-wide IRF5 binding sites using validated ChIP-grade antibodies

• Perturbation analysis: Monitor system-wide effects of IRF5 modulation using antibody readouts

• Dynamic studies: Use antibodies to track temporal changes in IRF5 expression and localization

Understanding IRF5's role as a transcriptional regulator requires comprehensive approaches that connect its expression and activation to downstream gene networks .

How can IRF5 antibodies help elucidate the role of IRF5 in COVID-19 pathogenesis?

IRF5 antibodies can contribute to COVID-19 research by:

• Expression analysis: Measuring IRF5 levels in COVID-19 patient samples versus controls

• Activation studies: Determining if SARS-CoV-2 infection activates IRF5

• Cell-type specific responses: Identifying which cells upregulate IRF5 during infection

• Intervention studies: Monitoring IRF5 changes in response to treatments

• Correlation with severity: Relating IRF5 expression/activation to disease severity

The search results indicate that IRF5 is a key transcription factor regulating the interferon response during SARS-CoV-2 infection , making IRF5 antibodies valuable tools for investigating COVID-19 immunopathology.

What are the most reliable protocols for IRF5 detection across different experimental contexts?

Based on the available research:

• Western blot: Cell Signaling #3257 (cs3257) has been validated for specific detection of human IRF5

• Immunoprecipitation: Clone 2E3-1A11 (WH0003663M1) has shown superior performance

• Flow cytometry: Antibodies should be validated with IRF5 knockout or knockdown controls

• Immunohistochemistry: Validation with appropriate controls is essential, though specific validated antibodies aren't identified in the search results

The research emphasizes that proper controls are crucial regardless of technique, and antibodies should be validated for each specific application .

How might future antibody technologies improve IRF5 research?

Emerging antibody technologies that could advance IRF5 research include:

• Proximity labeling antibodies: For studying IRF5 interactomes in specific cellular compartments

• Intrabodies: For tracking IRF5 in living cells

• Bispecific antibodies: For detecting IRF5 interactions with specific partners

• Conformation-specific antibodies: For distinguishing active versus inactive IRF5

• Degradation-inducing antibodies: For targeted IRF5 degradation without genetic manipulation

These approaches could provide more specific, dynamic, and functional insights into IRF5 biology beyond traditional antibody applications.

What are the most important considerations for reproducibility in IRF5 antibody-based experiments?

To ensure reproducibility:

• Complete antibody documentation: Report catalog numbers, clones, lots, and dilutions

• Control inclusion: Always include and report appropriate positive and negative controls

• Validation evidence: Provide evidence of antibody specificity testing

• Protocol standardization: Use standardized protocols with detailed methods

• Multiple antibody approach: Consider using multiple antibodies targeting different epitopes

• Independent verification: Confirm key findings using complementary approaches

The research highlights that many published studies may have used antibodies that cannot specifically detect IRF5, emphasizing the need for proper validation and reporting to improve reproducibility .