IREB2 Antibody

What is IREB2 and why is it important to study?

IREB2 is an RNA-binding protein that binds to iron-responsive elements (IREs) found in the untranslated regions of mRNAs encoding proteins involved in iron metabolism. When bound to the IRE element in ferritin mRNA's 5'-UTR, it represses translation. Conversely, when bound to transferrin receptor mRNA's 3'-UTR, it inhibits degradation of this otherwise rapidly degraded transcript .

IREB2 plays a crucial role in cellular iron homeostasis, with its regulation strongly controlled by iron availability – high iron levels trigger its degradation . Beyond iron metabolism, IREB2 has been implicated in hypoxia responses, neurodegenerative disorders, and COPD susceptibility, making it a significant target for diverse research areas . Recent studies have identified biallelic variants in IREB2 associated with early-onset neurodegenerative syndrome, highlighting its importance in neurological research .

What types of IREB2 antibodies are commercially available and how do they differ?

Commercial IREB2 antibodies are available in three main formats, each with distinct characteristics:

Commercial catalogs feature over 223 IREB2 antibodies from 26 suppliers, with varying applications, host species, and reactivity profiles . Research indicates that recombinant antibodies typically outperform both monoclonal and polyclonal antibodies in reproducibility and specificity .

What are the primary applications for IREB2 antibodies in research?

IREB2 antibodies support multiple experimental approaches in iron metabolism and disease research:

Researchers should note that antibody performance is application-specific, and validation should be performed for each experimental system .

How should I select an appropriate IREB2 antibody for my research?

When selecting an IREB2 antibody, consider these key factors:

• Experimental application: Choose antibodies validated for your specific application (WB, IHC, IF, etc.).

• Species reactivity: Ensure compatibility with your experimental model (human, mouse, rat, etc.).

• Epitope location: Consider whether targeting specific protein domains is relevant to your research question.

• Validation data: Prioritize antibodies with extensive validation, particularly those tested in knockout systems .

• Antibody type: Recombinant antibodies offer superior reproducibility compared to polyclonal or monoclonal options .

• Citation record: Antibodies with published research applications provide greater confidence in performance .

Review vendor datasheets for specificity information, validation methods, and recommended protocols. Cross-reference this information with published literature to evaluate antibody performance in contexts similar to your research .

What are the most rigorous methods for validating IREB2 antibody specificity?

Antibody validation is critical for ensuring experimental reproducibility. For IREB2 antibodies, the following hierarchical validation approaches are recommended:

• Genetic validation (Gold standard): Test antibody specificity in IREB2 knockout systems using CRISPR/Cas9 or RNAi approaches. A validated antibody should show no signal in knockout samples .

• Orthogonal validation: Compare antibody-based measurements with antibody-independent methods (e.g., mass spectrometry or RNA-seq) .

• Independent antibody validation: Use multiple antibodies targeting different IREB2 epitopes - concordant results increase confidence .

• Expression validation: Test antibody in systems with manipulated IREB2 expression (overexpression or knockdown) .

• Immunoprecipitation-mass spectrometry: Confirm that the antibody captures primarily IREB2 protein rather than off-target proteins .

Recent advances in validation technology emphasize the importance of knockout controls, particularly for Western blots and immunofluorescence applications. YCharOS studies found that knockout cell lines provide superior validation compared to other control types .

How can I address batch-to-batch variability when using IREB2 antibodies in longitudinal studies?

Batch-to-batch variability represents a significant challenge for reproducible research, particularly with polyclonal antibodies. Implement these strategies to mitigate variability:

• Prefer recombinant antibodies: When possible, use recombinant IREB2 antibodies, which demonstrate superior consistency compared to polyclonal alternatives .

• Bulk purchasing: Secure sufficient antibody from a single lot to complete your entire study.

• Lot validation: For each new lot, perform side-by-side validation with previous lots using identical samples and protocols.

• Internal standards: Include consistent positive and negative controls in each experiment to normalize across batches.

• Detailed record-keeping: Document lot numbers, dilutions, and experimental conditions for all antibody-based experiments.

• Bridging studies: When changing lots is unavoidable, perform comprehensive bridging studies to establish correlation factors between lots.

Research has shown that polyclonal antibodies exhibit particularly high lot-to-lot variability due to their complex composition of antibody populations, which can vary even with affinity purification .

What are the critical considerations when using IREB2 antibodies for studying iron-dependent regulation?

IREB2 protein levels and activity are highly sensitive to cellular iron status, presenting unique challenges when studying its regulation:

• Iron status standardization: Carefully control cellular iron status using defined media compositions and validated iron chelators or supplements.

• Fixation protocols: Iron manipulation can affect epitope accessibility. Optimize fixation conditions for each experimental paradigm.

• Timing considerations: IREB2 undergoes iron-dependent degradation, so standardize harvest times relative to iron manipulations.

• Subcellular localization: Consider that iron status may affect IREB2 subcellular distribution, potentially impacting antibody accessibility.

• Complementary approaches: Pair antibody detection with functional assays (RNA-binding activity) to correlate protein levels with functional status.

• Control proteins: Include detection of iron-insensitive proteins and known iron-responsive targets (ferritin, transferrin receptor) as internal controls.

When studying iron-dependent regulation, researchers should be aware that high iron levels lead to IREB2 degradation, which may affect detection sensitivity .

How should I approach epitope selection when studying IREB2 variants or post-translational modifications?

IREB2 function is regulated through complex mechanisms including post-translational modifications and variant-specific effects. Strategic epitope selection is critical:

• Domain awareness: IREB2 contains an iron-responsive domain and RNA-binding regions. Select antibodies targeting domains relevant to your research question.

• Modification-specific antibodies: For phosphorylation, ubiquitination, or other modifications, use antibodies specifically validated for modified forms.

• Variant considerations: When studying disease-associated variants (e.g., A1111G or A2477T), verify that antibody epitopes are not affected by the mutation .

• Epitope mapping: For critical applications, consider epitope mapping to precisely identify antibody binding regions.

• Recombinant controls: Express wild-type and variant IREB2 (as in the study using Flag-IREB2-WT, Flag-IREB2-A1111G constructs) to validate antibody recognition .

When studying specific IREB2 variants associated with neurodegenerative conditions, it's essential to confirm that your antibody can detect the variant form with comparable efficiency to wild-type protein .

What technical challenges might I encounter when performing co-immunoprecipitation with IREB2 antibodies?

Co-immunoprecipitation (Co-IP) studies with IREB2 present several technical challenges:

• RNA-dependent interactions: IREB2 functions as an RNA-binding protein, so distinguish direct protein-protein interactions from RNA-mediated associations by including RNase controls.

• Iron-dependent binding: IREB2 interactions may be influenced by iron status; standardize and report cellular iron conditions.

• Antibody interference: The antibody binding site may overlap with protein interaction domains; test multiple antibodies targeting different epitopes.

• Buffer optimization: RNA-protein complexes require specialized buffers - optimize salt concentration, detergents, and RNase inhibitors.

• Cross-linking considerations: For transient interactions, consider reversible cross-linking strategies, being mindful that they may affect epitope recognition.

• Validation approaches: Confirm interactions using reciprocal Co-IP and orthogonal methods like proximity ligation assays.

Several IREB2 antibodies have been validated for Co-IP applications, with monoclonal antibodies generally providing cleaner results due to reduced background binding .

What are the recommended protocols for IREB2 Western blotting?

Optimal Western blot detection of IREB2 requires specific technical considerations:

Sample Preparation:

• Use RIPA buffer supplemented with protease inhibitors and iron chelators (to prevent degradation).

• Include phosphatase inhibitors if studying phosphorylation status.

• Standardize protein loading to 20-50 μg per lane.

Protocol Recommendations:

• Use 6-8% gels for optimal separation of the ~105 kDa IREB2 protein.

• Transfer to PVDF membrane at 100V for 90 minutes (wet transfer recommended).

• Block with 5% non-fat milk in TBST for 1 hour at room temperature.

• Incubate with primary antibody at recommended dilution (typically 1:200-1:1000) .

• Wash thoroughly (4 × 5 minutes with TBST).

• Use appropriate HRP-conjugated secondary antibody.

• Develop using enhanced chemiluminescence.

Troubleshooting Tips:

• If detecting multiple bands, note that an additional band at ~60 kDa has been observed with some antibodies .

• Multiple bands may represent degradation products or isoforms; include positive controls.

• For weak signals, extend primary antibody incubation to overnight at 4°C.

• For high background, increase blocking time or washing steps.

How can I optimize immunohistochemistry protocols for IREB2 detection in tissue samples?

Successful IHC detection of IREB2 requires careful optimization:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours.

• Process and embed in paraffin following standard protocols.

• Section at 4-5 μm thickness.

Antigen Retrieval Options:

• Primary recommendation: TE buffer (pH 9.0), heat-induced epitope retrieval for 20 minutes .

• Alternative method: Citrate buffer (pH 6.0) may be used if TE buffer yields suboptimal results .

Staining Protocol:

• Block endogenous peroxidase (3% H₂O₂, 10 minutes).

• Block non-specific binding (10% normal serum, 1 hour).

• Apply primary antibody at optimized dilution (1:50-1:500) .

• Incubate overnight at 4°C.

• Apply appropriate detection system (e.g., HRP-polymer).

• Develop with DAB and counterstain with hematoxylin.

Validation Considerations:

• Include positive control tissues (spleen, colon) .

• Include negative controls (primary antibody omission, isotype controls).

• Consider dual staining with iron markers for co-localization studies.

What controls are essential when using IREB2 antibodies to study iron-responsive cellular mechanisms?

Robust controls are crucial when studying IREB2's role in iron homeostasis:

Essential Controls:

• Iron Manipulation Controls:

  • Positive control: Cells treated with iron chelators (e.g., DFO) to increase IREB2 levels

  • Negative control: Cells treated with iron (e.g., ferric ammonium citrate) to decrease IREB2 levels

• Genetic Controls:

  • IREB2 knockdown/knockout samples

  • IREB2 overexpression systems (wild-type and mutant constructs)

• Specificity Controls:

  • Secondary antibody-only controls

  • Isotype controls

  • Competing peptide blocking (if available)

• Functional Controls:

  • Detection of known IREB2-regulated targets (ferritin, transferrin receptor)

  • RNA-binding assays to correlate protein levels with activity

• Technical Controls:

  • Loading controls (β-actin, GAPDH) for Western blots

  • Tissue/cell type-specific markers for IHC/IF

When studying iron-related mechanisms, it's critical to verify that observed changes in IREB2 levels reflect physiological responses rather than technical artifacts .

How should I approach troubleshooting when IREB2 antibodies yield inconsistent results?

When facing inconsistent results with IREB2 antibodies, implement this systematic troubleshooting approach:

Step 1: Antibody Validation

• Verify antibody specificity using positive and negative controls

• Check if the antibody has been validated for your specific application

• Review recent literature for reported issues with the specific antibody

Step 2: Technical Optimization

• Titrate antibody concentration using a dilution series

• Optimize incubation conditions (time, temperature)

• Test different blocking agents to reduce background

• For Western blots, try different membrane types and transfer conditions

Step 3: Sample Preparation Assessment

• Ensure consistent sample collection and processing

• Verify protein integrity with total protein stains

• Consider fresh sample preparation if degradation is suspected

• For tissue samples, optimize fixation and antigen retrieval

Step 4: Alternative Approaches

• Test different antibodies targeting distinct IREB2 epitopes

• Consider alternative detection methods (fluorescent vs. chemiluminescent)

• Implement orthogonal detection methods (mass spectrometry, RNA analysis)

Step 5: Environmental Variables

• Control for iron status in culture media or tissue preparation

• Standardize harvest times and cell confluency

• Document lot numbers and storage conditions

Research has shown that approximately 50% of commercial antibodies fail to meet basic standards for characterization, making troubleshooting a critical skill for researchers .

How can IREB2 antibodies be effectively used in multiplex imaging applications?

Multiplex imaging of IREB2 with other iron metabolism markers provides valuable insights into co-regulation patterns:

Optimization Strategies:

• Antibody selection: Choose antibodies raised in different host species to enable simultaneous detection.

• Sequential staining: For same-species antibodies, consider sequential staining with stripping steps.

• Spectral unmixing: Use fluorophores with minimal spectral overlap to reduce bleed-through.

• Signal amplification: For low-abundance targets, implement tyramide signal amplification.

• Automation: Consider automated staining platforms for consistent multiplex results.

Recommended Multiplex Panels:

• IREB2 + Ferritin + Transferrin receptor (iron metabolism panel)

• IREB2 + Cellular compartment markers (nuclear, cytoplasmic, endosomal)

• IREB2 + Cell-type markers (neurons, glia, macrophages)

Validation Approaches:

• Single-stain controls to establish baseline signals

• Fluorophore minus one (FMO) controls to assess spectral overlap

• Quantitative colocalization analysis with appropriate statistical metrics

When designing multiplex panels, carefully consider fixation conditions, as they may affect epitope accessibility differently for each target protein .

What are the emerging applications of IREB2 antibodies in neurodegenerative disease research?

IREB2 has emerging significance in neurodegenerative disease research, with several promising applications:

• Genetic Neurodegeneration Models: Recently identified biallelic variants in IREB2 cause early-onset neurodegenerative syndrome, creating opportunities to study disease mechanisms using antibody-based approaches .

• COPD-Neurodegeneration Connection: IREB2 has been implicated in COPD susceptibility, with potential overlap to neurological manifestations, enabling investigation of shared pathological mechanisms .

• Iron Dyshomeostasis: Neurodegenerative diseases often feature disrupted iron metabolism. IREB2 antibodies enable visualization of iron regulatory networks in patient samples.

• Therapeutic Target Validation: As iron chelation therapies advance in neurodegeneration, IREB2 antibodies provide tools to monitor target engagement and pathway modulation.

• Biomarker Development: Changes in IREB2 expression or localization may serve as disease progression markers, assessable via antibody-based assays.

Researchers should consider paired analysis of wild-type and mutant IREB2 (e.g., A1111G, A2477T variants) to elucidate pathological mechanisms .

What are the best practices for integrating IREB2 antibody data with other omics approaches?

Multi-omics integration with IREB2 antibody data requires careful experimental design:

Integration Strategies:

• Proteogenomic Correlation:

  • Correlate IREB2 protein levels (antibody-based) with mRNA expression (RNA-seq)

  • Identify post-transcriptional regulation by comparing protein:mRNA ratios

  • Map antibody binding sites to specific transcript isoforms

• Functional Genomics Validation:

  • Confirm antibody specificity in CRISPR-edited cell lines

  • Use antibodies to validate genomic findings from GWAS studies (e.g., IREB2 in COPD)

  • Correlate genotype with protein expression patterns

• Multi-scale Imaging Integration:

  • Combine antibody-based microscopy with spatial transcriptomics

  • Correlate subcellular localization with interactome data

  • Implement machine learning approaches to identify pattern correlations

• Temporal Dynamics Analysis:

  • Design time-course experiments with parallel antibody-based and omics measurements

  • Account for different temporal responses between transcript and protein

  • Develop mathematical models to predict protein changes from transcriptomic data

When integrating across platforms, standardize sample processing and implement robust normalization strategies to minimize technical variation .

How should researchers address reproducibility concerns with IREB2 antibodies in publication?

To enhance reproducibility and transparency in IREB2 antibody research:

Reporting Standards:

• Detailed Antibody Documentation:

  • Report complete antibody information: supplier, catalog number, lot number, RRID

  • Specify antibody type (polyclonal, monoclonal, recombinant)

  • Document immunogen information and target epitope when available

• Validation Evidence:

  • Include validation data within publications or supplementary materials

  • Describe validation approach (genetic, orthogonal, multiple antibodies)

  • Report negative results from antibody testing

• Protocol Transparency:

  • Provide complete methodological details (dilutions, incubation times, buffers)

  • Share unprocessed original images alongside processed data

  • Consider protocol registration or deposition in repositories

• Control Documentation:

  • Describe all controls used (positive, negative, technical)

  • Include representative images of controls

  • Quantify signal-to-background ratios

• Data Availability:

  • Deposit raw image data in appropriate repositories

  • Consider antibody validation datasets as valuable research outputs

These practices address the estimated $0.4-1.8 billion annual losses in the United States alone due to poorly characterized antibodies .

What recent technological advances are improving IREB2 antibody performance and validation?

Several technological advances are enhancing IREB2 antibody quality and validation:

• Recombinant Antibody Production: Sequence-defined recombinant antibodies offer superior reproducibility compared to traditional hybridoma-derived monoclonals or polyclonals .

• CRISPR-based Validation: Genome-edited cell lines provide gold-standard negative controls for antibody validation, particularly valuable for IREB2 where specificity is crucial .

• Automated Characterization Platforms: High-throughput platforms like those used by YCharOS enable comprehensive antibody testing across multiple applications and conditions .

• Machine Learning Applications: AI algorithms help identify optimal antibody candidates from libraries and predict cross-reactivity.

• Nanobody Technology: Single-domain antibodies offer improved access to conformational epitopes and reduced steric hindrance in complex samples.

• Open Science Initiatives: Community resources like NeuroMab and NABOR are improving antibody quality and accessibility through transparent validation and sequence sharing .

• Spatial Proteomics Integration: Advanced microscopy combined with antibody-based detection enables subcellular localization studies with unprecedented resolution.

Research indicates that antibodies validated using these advanced approaches significantly outperform traditional antibodies, with recombinant antibodies showing particularly strong performance across multiple applications .