ELF3 Antibody

What is ELF3 and why is it an important research target?

ELF3 (E74-Like Factor 3), also known as ESE-1, ESX, ERT, EPR-1, and JEN, is a 41-43 kDa member of the ETS family of transcription factors. It plays a crucial role in epithelial cell differentiation during normal homeostasis by repressing genes needed during early differentiation and promoting genes required for terminal differentiation. During inflammatory conditions, ELF3 expression extends beyond epithelial cells to include monocytes, endothelial cells, and chondrocytes, where it regulates the production of molecules such as Ang1 and COX2 . As a transcription factor with context-dependent functions, ELF3 has become an important target in studies of epithelial development, inflammation, and cancer progression.

Which applications are most commonly supported by ELF3 antibodies?

ELF3 antibodies support numerous experimental applications, with Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) being the most widely validated. Many antibodies also support ELISA, Immunocytochemistry (ICC), Immunoprecipitation (IP), and Chromatin Immunoprecipitation (ChIP) . When selecting an ELF3 antibody, researchers should verify that it has been validated for their specific application, as performance can vary significantly between techniques. For particularly sensitive applications like ChIP-seq, specialized antibodies like Cell Signaling Technology's ELF3 (F8J2G) Rabbit mAb have been specifically validated .

What sample types are compatible with ELF3 antibodies?

ELF3 antibodies have been validated with various sample types including:

• Cell lysates from multiple cell lines (Jurkat, PC-3, HT-29, EL-4, A549)

• Paraffin-embedded tissue sections (e.g., human prostate)

• Recombinant proteins for quantitative assays

For optimal results, sample preparation protocols should be tailored to both the antibody specifications and the experimental application. Heat-induced epitope retrieval using basic antigen retrieval reagents has been successfully employed for paraffin-embedded tissues prior to IHC with ELF3 antibodies .

What controls should be included in experiments using ELF3 antibodies?

For rigorous experimental design with ELF3 antibodies, researchers should include:

• Positive controls: Cell lines with known ELF3 expression (e.g., HT-29, PC-3, A549)

• Negative controls: Samples where primary antibody is omitted

• Specificity controls: Ideally, ELF3 knockout/knockdown samples

• Isotype controls: Matching IgG from the same species as the primary antibody

• Loading controls: For quantitative Western blot analysis

Some antibodies, such as R&D Systems' Human ELF3 Antibody (MAB57871), have been validated using knockout/knockdown approaches, providing higher confidence in their specificity .

How can researchers optimize ELF3 detection in different cell and tissue types?

Optimizing ELF3 detection requires consideration of its context-dependent expression patterns:

• Epithelial tissues: Use heat-induced epitope retrieval with basic pH buffers (e.g., Antigen Retrieval Reagent-Basic) for paraffin sections . Nuclear staining is expected in glandular epithelial cells.

• Inflammatory contexts: When examining ELF3 in non-epithelial cells during inflammation, consider dual staining with cell-type specific markers (e.g., CD68 for macrophages) to confirm identity of ELF3-expressing cells.

• Cancer cell lines: Different cancer lines show variable ELF3 expression levels. A549, HT-29, and PC-3 lines are reliable positive controls for human samples , while EL-4 serves as a mouse control.

• Antibody concentration optimization: For IHC, starting concentrations around 15 μg/mL have been effective , but titration experiments should be performed for each new tissue type or experimental system.

• Detection system selection: For low abundance detection, consider using amplification-based detection systems such as HRP-DAB rather than direct fluorescence methods.

What are the major challenges in interpreting ELF3 antibody experimental results?

Interpreting ELF3 antibody results presents several challenges:

• Multiple protein isoforms: ELF3 can exist in multiple forms, with the main form at approximately 42 kDa . Unexpected bands may represent legitimate isoforms rather than non-specific binding.

• Cross-reactivity with ETS family members: The ETS family shares conserved DNA-binding domains. While some antibodies have been tested for cross-reactivity (e.g., no cross-reactivity with ELF5 for certain clones) , comprehensive cross-reactivity profiles are often unavailable.

• Context-dependent expression: ELF3 expression changes dramatically during inflammation or disease states, so interpreting expression changes requires careful consideration of the physiological context.

• Nuclear vs. cytoplasmic localization: As a transcription factor, ELF3 is typically nuclear, but cytoplasmic localization can occur. Discrepancies in localization patterns between studies may reflect biological differences rather than antibody performance issues.

• Signal intensity interpretation: Quantitative comparisons of ELF3 levels between different studies should be approached with caution due to variations in antibody affinity, detection methods, and exposure settings.

How do monoclonal and polyclonal ELF3 antibodies compare in research applications?

The choice between monoclonal and polyclonal ELF3 antibodies significantly impacts experimental outcomes:

For the most critical experiments, researchers may benefit from comparing results with both monoclonal and polyclonal antibodies to ensure robust findings.

What technical approaches can resolve contradictory ELF3 antibody data between studies?

When faced with contradictory results between studies using ELF3 antibodies, researchers should consider these resolution approaches:

• Antibody validation comparison: Assess whether antibodies were validated by knockout/knockdown controls . Prioritize findings from studies using comprehensively validated antibodies.

• Epitope mapping: Different antibodies recognize distinct regions of ELF3 (e.g., N-terminal region vs. full-length protein) . These differences may explain discrepancies if protein processing occurs in certain contexts.

• Multi-antibody verification: Confirm key findings using multiple antibodies targeting different ELF3 epitopes. Concordant results with diverse antibodies increase confidence.

• Orthogonal techniques: Complement antibody-based detection with non-antibody methods (e.g., mass spectrometry, RNA analysis) to independently verify findings.

• Recombinant ELF3 controls: Use recombinant human ELF3 proteins (e.g., Met1-Gly173 fragment) as standards to calibrate detection sensitivity across studies.

• Protocol standardization: Implement identical sample preparation, antibody concentration, and detection methods to determine if methodological differences explain contradictory results.

How should researchers select the optimal ELF3 antibody for their specific experimental needs?

Selection of the optimal ELF3 antibody should follow this systematic approach:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF, ChIP, etc.) .

• Species reactivity: Confirm reactivity with your species of interest. Some antibodies react with human ELF3 only, while others cross-react with mouse and rat orthologs .

• Epitope consideration: For mechanistic studies, select antibodies targeting functional domains relevant to your hypothesis. N-terminal (Met1-Gly173) targeting antibodies are common .

• Validation rigor: Prioritize antibodies validated by knockout/knockdown approaches over those validated only by overexpression systems.

• Clonality decision: For quantitative studies requiring high reproducibility, monoclonal antibodies offer advantages. For initial detection in complex samples, polyclonal antibodies may provide higher sensitivity.

• Format requirements: Consider whether you need unconjugated antibodies or specific conjugates (HRP, fluorophores) based on your detection system.

• Literature precedent: Select antibodies with published track records in experimental systems similar to yours.

What are effective troubleshooting strategies for weak or non-specific ELF3 staining?

When encountering issues with ELF3 antibody performance, consider these troubleshooting approaches:

For weak signal:

• Antibody concentration: Increase primary antibody concentration incrementally (e.g., from 1 μg/mL to 5-15 μg/mL) .

• Epitope retrieval optimization: For IHC/IF, test different antigen retrieval methods and durations. Basic pH buffers have proven effective for some ELF3 antibodies .

• Incubation conditions: Extend primary antibody incubation time (overnight at 4°C) or adjust temperature.

• Detection system enhancement: Switch to more sensitive detection systems (e.g., tyramide signal amplification).

• Sample preparation review: Ensure sample processing maintains ELF3 integrity (avoid excessive fixation for tissues).

For non-specific signal:

• Blocking optimization: Increase blocking buffer concentration or duration.

• Antibody specificity verification: Test the antibody on known negative controls or ELF3 knockout samples .

• Wash stringency increase: Add additional wash steps or detergents to reduce non-specific binding.

• Secondary antibody cross-reactivity check: Test secondary antibody alone to rule out non-specific binding.

• Cross-adsorption: For polyclonal antibodies with cross-reactivity issues, consider custom cross-adsorption against potential cross-reacting proteins.

How can researchers quantitatively compare ELF3 expression across different experimental conditions?

For rigorous quantitative analysis of ELF3 expression:

• Western blot quantification:

  • Use recombinant ELF3 standards to create a standard curve

  • Ensure linear dynamic range of detection

  • Normalize to appropriate loading controls

  • Apply consistent image acquisition settings across all samples

  • Use technical replicates (minimum triplicate) for statistical analysis

• IHC/IF quantification:

  • Implement standardized scoring systems (H-score, Allred score)

  • Use digital image analysis with consistent thresholds

  • Include reference standards in each batch

  • Blind scorers to experimental conditions

  • Quantify multiple fields per sample (minimum 5-10)

• ELISA-based quantification:

  • Develop sandwich ELISA using validated ELF3 antibody pairs

  • Include recombinant ELF3 standard curves

  • Process all samples simultaneously when possible

  • Account for matrix effects in complex samples

• Flow cytometry:

  • Use isotype controls and fluorescence-minus-one controls

  • Report relative fluorescence intensity or molecules of equivalent soluble fluorochrome

  • Standardize using calibration beads between experiments

• Controls for normalization:

  • Include identical reference samples across all experimental batches

  • Consider normalizing to total protein rather than single housekeeping proteins

  • Document and report all normalization methods in detail

What is the significance of ELF3 subcellular localization in experimental interpretation?

ELF3's subcellular localization provides critical functional insights:

• Nuclear localization: As a transcription factor, nuclear ELF3 typically indicates active transcriptional regulation . Predominantly observed in:

  • Differentiated epithelial cells

  • Nuclei of glandular epithelial cells in prostate tissue

  • Cancer cells with active ELF3-dependent transcriptional programs

• Cytoplasmic localization: May indicate:

  • Sequestration as a regulatory mechanism

  • Post-translational modifications affecting nuclear import

  • Potential non-transcriptional functions

  • Pathological states in certain disease contexts

• Dual localization patterns: Often observed during dynamic cellular processes:

  • Developmental transitions

  • Inflammatory responses

  • Early stages of malignant transformation

• Methodological considerations for localization studies:

  • Fixation methods significantly impact observed localization

  • Cell fractionation coupled with Western blotting provides quantitative assessment

  • Co-localization with compartment markers enhances interpretative value

  • Live-cell imaging with fluorescently-tagged ELF3 can reveal dynamic localization changes

• Experimental controls for localization studies:

  • Include known nuclear proteins (e.g., HDAC1) and cytoplasmic proteins (e.g., GAPDH)

  • Verify antibody access to different compartments using permeabilization controls

  • Consider dual staining approaches with organelle-specific markers

How do ELF3 antibodies contribute to cancer research?

ELF3 antibodies have become valuable tools in cancer research across multiple applications:

• Diagnostic biomarker development:

  • IHC detection of ELF3 in tissue microarrays helps evaluate expression across cancer types

  • Nuclear localization patterns in prostate cancer may have prognostic implications

  • Quantitative analysis of ELF3 levels correlates with clinical outcomes in some epithelial cancers

• Mechanistic studies:

  • ChIP and ChIP-seq using ELF3 antibodies reveal target genes in cancer progression

  • Co-immunoprecipitation identifies protein interaction partners in cancer-specific contexts

  • Phospho-specific ELF3 antibodies detect activation states

• Therapeutic target validation:

  • Antibody-based detection confirms target engagement in drug development

  • Monitoring ELF3 levels during treatment response

  • Validation of ELF3 knockdown/knockout models

• Cancer cell line characterization:

  • Western blot analysis reveals variable ELF3 expression across cancer cell lines

  • Detected in Jurkat (T cell leukemia), PC-3 (prostate cancer), HT-29 (colon adenocarcinoma), and A549 (lung carcinoma) lines

  • Correlation between ELF3 levels and phenotypic characteristics

• Cancer-inflammation interface:

  • Dual staining with inflammatory markers reveals ELF3's role at the intersection of inflammation and cancer

What advances in ELF3 antibody technology are improving research capabilities?

Recent technological advances are enhancing ELF3 antibody applications:

• Antibody engineering improvements:

  • Recombinant antibody technology ensures batch-to-batch consistency

  • Single B cell cloning approaches yield antibodies with exceptional specificity

  • Humanized antibodies enable in vivo applications with reduced immunogenicity

• Validation enhancements:

  • Knockout validation using CRISPR/Cas9-edited cell lines

  • Multi-epitope targeting strategies reduce false positive/negative results

  • Comprehensive cross-reactivity testing against related ETS family members

• Application-specific optimizations:

  • ChIP-seq grade antibodies with validated performance in genome-wide applications

  • Super-resolution microscopy compatible antibodies for nanoscale localization

  • Multiplexed detection systems for co-localization with other biomarkers

• Novel formats and conjugates:

  • Bifunctional antibodies for proximity ligation assays

  • Site-specific conjugation methods preserving antigen-binding capacity

  • Nanobody and aptamer alternatives for applications requiring smaller binding molecules

• Reproducibility initiatives:

  • Standardized reporting of validation parameters

  • Antibody registry entries with unique identifiers

  • Independent validation through antibody validation initiatives

How can ELF3 antibodies be used to study inflammatory processes?

ELF3 antibodies provide valuable insights into inflammatory mechanisms:

• Cell type-specific expression analysis:

  • Detect ELF3 induction in non-epithelial cells during inflammation

  • Monitor expression in monocytes, endothelial cells, and chondrocytes during inflammatory states

  • Quantify temporal expression patterns during inflammatory progression

• Signaling pathway investigations:

  • Combine with phospho-specific antibodies against inflammatory mediators

  • ChIP analysis of ELF3 binding to inflammatory gene promoters

  • Co-localization with nuclear translocation of NF-κB and other inflammatory transcription factors

• Inflammatory mediator regulation:

  • Track ELF3-dependent expression of Ang1 and COX2

  • Monitor ELF3 binding to inflammatory gene promoters via ChIP

  • Correlate ELF3 levels with inflammatory cytokine production

• Tissue-specific inflammatory responses:

  • Compare ELF3 patterns across multiple inflamed tissues

  • Evaluate epithelial-specific vs. immune cell-specific ELF3 functions

  • Study interface between epithelial damage and inflammatory cell recruitment

• Chronic inflammation models:

  • Track ELF3 expression during transition from acute to chronic inflammation

  • Correlate with tissue remodeling and fibrosis markers

  • Evaluate as potential biomarker for inflammatory disease progression

What considerations are important when designing longitudinal studies that rely on ELF3 antibodies?

Longitudinal studies using ELF3 antibodies require special considerations:

• Antibody stability and storage:

  • Aliquot antibodies to minimize freeze-thaw cycles

  • Store at recommended temperatures (-20°C to -70°C for long-term, 2-8°C for 1 month after reconstitution)

  • Document lot numbers and purchase sufficient quantity from single lots

• Protocol standardization:

  • Establish detailed standard operating procedures

  • Maintain consistent antibody concentrations (e.g., 1 μg/mL for Western blot)

  • Use identical buffer compositions throughout the study

  • Process samples using consistent methods and timing

• Instrument calibration:

  • Regular calibration of imaging systems and readers

  • Include calibration standards in each experimental batch

  • Document all instrument settings for reproducibility

• Reference standards:

  • Prepare master aliquots of positive control samples

  • Include recombinant ELF3 standards at multiple concentrations

  • Create standard curves for quantitative analyses

• Data normalization strategies:

  • Establish baseline ELF3 levels for each subject/sample

  • Use relative rather than absolute quantification when comparing across time points

  • Include time-matched controls for each experimental time point

  • Account for batch effects in statistical analyses

What experimental designs best capture the dual role of ELF3 in epithelial differentiation and inflammation?

Optimal experimental designs to study ELF3's dual functions include:

• Temporal monitoring systems:

  • Time-course analysis of ELF3 expression during epithelial differentiation

  • Parallel tracking of differentiation markers and inflammatory mediators

  • Sequential sampling during inflammatory challenge and resolution

• Cell type-specific approaches:

  • Co-staining with epithelial markers (E-cadherin, cytokeratins) and inflammatory cell markers

  • Cell sorting followed by ELF3 protein quantification

  • Single-cell analysis correlating ELF3 levels with cell phenotypes

• Conditional expression systems:

  • Inducible ELF3 expression in epithelial and non-epithelial cells

  • Tissue-specific knockout models evaluated under homeostatic and inflammatory conditions

  • Domain mutant expression to separate differentiation vs. inflammatory functions

• 3D culture models:

  • Epithelial organoids with inflammatory cell co-culture

  • Air-liquid interface cultures with inflammatory stimulation

  • Wound healing models to capture transition states

• Integrative analysis approaches:

  • Combined ChIP-seq and RNA-seq to identify context-dependent gene regulation

  • Proteomics to identify interaction partners in different cellular contexts

  • Multi-omics integration to map ELF3 function across biological states

How can researchers effectively combine ELF3 antibodies with other molecular tools for comprehensive mechanistic studies?

Integrated experimental approaches combining ELF3 antibodies with complementary tools:

• Multi-omic strategies:

  • ChIP-seq with ELF3 antibodies combined with RNA-seq and ATAC-seq

  • Proteomics following ELF3 immunoprecipitation

  • Integration with DNA methylation and histone modification data

• Functional genomics integration:

  • CRISPR-Cas9 modification of ELF3 binding sites

  • Correlation of ELF3 binding (ChIP) with functional enhancer assays

  • ELF3 knockout paired with rescue experiments using domain mutants

• Live-cell dynamics:

  • Combine fixed-cell antibody staining with live-cell ELF3-fluorescent protein fusions

  • FRAP (Fluorescence Recovery After Photobleaching) to assess dynamics, validated by antibody staining

  • Optogenetic ELF3 control systems with antibody-based outcome measurement

• Protein interaction networks:

  • Proximity labeling (BioID, APEX) with ELF3 fusions, validated by co-immunoprecipitation

  • Protein complementation assays combined with antibody-based localization

  • Mammalian two-hybrid screens with ELF3 domains, confirmed by co-immunoprecipitation

• Translational approaches:

  • Patient-derived xenografts with human-specific ELF3 antibody detection

  • Ex vivo tissue culture with pharmacological manipulation and ELF3 monitoring

  • Correlation of genomic alterations with antibody-detected ELF3 protein levels