ELF3 Antibody, HRP conjugated

What are the optimal conditions for using ELF3 antibody, HRP conjugated in Western blotting?

For optimal Western blotting with ELF3 antibody, HRP conjugated, researchers should consider the following protocol parameters:

The recommended dilution for Western blotting applications is typically 1:1000, though this may need optimization based on your specific antibody lot and experimental system . ELF3 is detected at approximately 42 kDa under reducing conditions, and blotting should be performed using appropriate buffer systems such as Immunoblot Buffer Group 1 .

When establishing your protocol, consider these critical steps:

• Use PVDF membrane rather than nitrocellulose for better protein retention

• Include positive control lysates such as A431, PC-3, or A549 human cell lines, all of which express detectable levels of ELF3

• Run your gel under reducing conditions to ensure proper protein denaturation

• Include loading controls appropriate for your experimental system

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

Detection efficiency can be validated by examining known ELF3-expressing cell lines. In particular, PC-3 (prostate cancer), A549 (lung carcinoma), and NIH-3T3 (mouse embryonic fibroblast) cell lines have demonstrated reliable ELF3 detection in Western blotting .

How should I validate the specificity of my ELF3 antibody in different experimental systems?

Validating antibody specificity is crucial for generating reliable data. For ELF3 antibody validation, incorporate these approaches:

Genetic validation approaches:

• Compare staining patterns in ELF3 knockdown/knockout versus wild-type samples using siRNA or CRISPR-Cas9 technology

• Examine ELF3 overexpression systems (such as HBEC-KT cells with stable ELF3 overexpression) alongside controls to confirm signal enhancement

• Use available ELF3-null cell lines as negative controls if available

Technical validation methods:

• Perform peptide competition assays to confirm epitope-specific binding

• Compare staining patterns across multiple ELF3 antibodies targeting different epitopes

• Include isotype controls to rule out non-specific binding

• Verify tissue expression patterns match known ELF3 distribution (higher in epithelial tissues)

Researchers should note that ELF3 displays both nuclear and cytoplasmic localization in human tumors and cell lines, which may affect interpretation of specificity tests . Validation tests should account for this dual localization pattern.

What are the recommended sample preparation procedures for detecting ELF3 in immunohistochemistry?

For successful immunohistochemical detection of ELF3, consider these sample preparation guidelines:

When using paraffin-embedded tissue sections, heat-induced epitope retrieval is essential for optimal ELF3 detection. The recommended protocol includes:

• Use of Antigen Retrieval Reagent-Basic (such as pH 9.0 Tris-EDTA buffer)

• Incubation with primary ELF3 antibody at 3-10 μg/mL concentrations overnight at 4°C

• Detection using appropriate visualization systems such as HRP-DAB Cell & Tissue Staining Kit

• Counterstaining with hematoxylin to provide tissue context

For cell lines, the following approach has proven effective:

• Fixation of cells with 4% paraformaldehyde

• Permeabilization with 0.1% Triton X-100

• Blocking with appropriate serum (typically 5-10% normal serum from the species of secondary antibody origin)

• Incubation with ELF3 antibody at 1:50-1:200 dilution for immunohistochemistry applications

What patterns of ELF3 expression should I expect across different cell and tissue types?

ELF3 expression varies significantly across tissues and cancer types, which is crucial for interpreting staining results:

Expected tissue expression patterns:

• Strong expression in epithelial tissues, particularly in lung, liver, and breast

• Detectable expression in human liver paraffin sections with appropriate retrieval methods

• Variable expression in cancer cell lines; consistently detected in epithelial carcinoma lines (A431, PC-3, A549)

Cancer-specific expression:

• Significantly higher expression in BRCA1-associated breast tumors compared to non-BRCA1-associated tumors

• Elevated expression in basal-like breast cancer subtypes compared to other breast cancer molecular subtypes

• Frequently amplified in lung adenocarcinoma (LUAD) but not in lung squamous cell carcinoma (LUSC)

Understanding these expression patterns is critical for properly interpreting staining results and selecting appropriate positive controls for your specific research context.

How can I troubleshoot weak or nonspecific signals when using ELF3 antibody in my experiments?

When encountering challenges with ELF3 antibody staining, consider these troubleshooting approaches:

For weak or absent signals:

• Optimize antibody concentration - try a range from 1:50 to 1:1000 depending on application

• Enhance epitope retrieval - extend heating time or try different pH buffers (acidic vs. basic)

• Increase incubation time (overnight at 4°C often yields better results than shorter incubations)

• Use signal amplification systems specifically compatible with HRP-conjugated antibodies

• Verify sample preparation protocol to ensure protein integrity is maintained

For high background or nonspecific staining:

• Increase blocking time and concentration (5-10% normal serum or BSA)

• Include 0.1-0.3% Triton X-100 in wash buffers to reduce nonspecific membrane binding

• Test different fixation methods that may better preserve ELF3 epitopes

• Reduce primary antibody concentration

• Include additional washing steps between incubations

Researchers should note that ELF3 has dual localization patterns (nuclear and cytoplasmic), which may initially appear as nonspecific staining . Careful analysis with appropriate controls is necessary to distinguish genuine signal distribution from background.

What considerations are important when comparing ELF3 expression across different experimental models?

When comparing ELF3 expression across models, researchers should consider these factors:

Tissue and cell-type specificity:

• ELF3 functions are highly context-dependent and tissue-specific

• Expression patterns differ significantly between epithelial and non-epithelial tissues

• Cancer subtypes show differential ELF3 expression (e.g., higher in LUAD vs. LUSC)

Technical standardization requirements:

• Use identical sample preparation, antibody concentrations, and detection methods across all compared samples

• Process and image all samples simultaneously when possible

• Include calibration standards or common reference samples

• Normalize expression data to appropriate housekeeping genes or proteins (GAPDH or β-actin have been validated for ELF3 studies)

Genetic context considerations:

• KRAS or EGFR mutation status may influence ELF3 expression in lung cancer models

• ELF3 expression should be interpreted in context of BRCA1 status in breast cancer models

• Consider epithelial-to-mesenchymal transition (EMT) status, as ELF3 correlates with epithelial markers like E-cadherin and shows anti-correlation with EMT-promoting factors like ZEB1

How can I investigate the dual roles of ELF3 as both a tumor suppressor and oncogene in my research?

Investigating ELF3's context-dependent functions requires sophisticated experimental approaches:

Recommended experimental strategies:

• Develop tissue-specific conditional ELF3 knockout/knockin models to assess tissue-specific functions

• Employ isogenic cell line pairs with and without ELF3 expression to isolate its effects

• Use doxycycline-inducible ELF3 expression systems to study dose-dependent effects

• Compare ELF3 function across multiple cancer subtypes in parallel experiments

Research has demonstrated that ELF3 exhibits oncogenic properties in lung adenocarcinoma but tumor-suppressive functions in other epithelial cancers . This duality can be investigated through:

• Analysis of ELF3 binding partners and transcriptional targets in different cellular contexts

• Correlation of ELF3 expression with proliferation markers, invasion assays, and in vivo tumor growth

• Examination of genetic and epigenetic alterations of the ELF3 locus (1q32.1) across cancer types

• Investigation of post-translational modifications that might alter ELF3 function

In non-malignant bronchial epithelial cells (HBEC-KT), ELF3 overexpression increased proliferation but was insufficient for complete cellular transformation, suggesting context-dependent requirements for additional oncogenic events .

What methods are optimal for studying ELF3's protein-protein interactions and transcriptional networks?

To elucidate ELF3's interactome and gene regulatory networks, these methodologies are recommended:

For protein-protein interaction studies:

• Co-immunoprecipitation using ELF3 antibodies to identify interaction partners

• Proximity ligation assays to verify interactions in situ

• BiFC (Bimolecular Fluorescence Complementation) for live-cell interaction visualization

• Mass spectrometry following ELF3 pulldown to identify novel interaction partners

Research has identified several key ELF3 protein-protein interactions across cancer models including ERBB2, ERBB3, ETS1, TIMP3, ARHGEF6, CLDN4, and ZEB1 . Many of these interactions are deregulated in cancer contexts.

For transcriptional network studies:

• ChIP-seq using ELF3 antibodies to map genome-wide binding sites

• RNA-seq following ELF3 modulation to identify regulated genes

• ATAC-seq to identify chromatin accessibility changes dependent on ELF3

• Combinatorial analysis with other transcription factors, particularly in the context of super-enhancers

Studies have shown that inhibition of super-enhancer-associated targets (BRD4, EP300, CDK7) reduced ELF3 expression in cancer models, suggesting ELF3 is regulated by super-enhancer mechanisms . ELF3 also participates in complex transcriptional networks with EHF and TGIF1 in lung adenocarcinoma .

How can I explore the relationship between ELF3 and BRCA1 in breast cancer research?

The emerging connection between ELF3 and BRCA1 presents important research opportunities:

Experimental approaches to investigate ELF3-BRCA1 relationship:

• Analyze correlation between ELF3 and BRCA1 expression in clinical samples

• Examine effects of BRCA1 knockdown/knockout on ELF3 expression and vice versa

• Investigate mechanisms of ELF3 upregulation in BRCA1-deficient contexts

• Study effects of ELF3 modulation on genomic stability in BRCA1-deficient cells

Research has revealed that ELF3 expression is significantly higher in BRCA1-associated breast tumors than in non-BRCA1-associated breast tumors, with a negative correlation between BRCA1 and ELF3 expression levels . Furthermore, ELF3 shows highest expression in basal-like breast cancer, the predominant subtype in BRCA1 mutation carriers .

ELF3 appears to help suppress excessive genomic instability and promote transformation in the context of BRCA1 deficiency, suggesting a complex relationship that may be exploited for therapeutic purposes .

How should I interpret differential subcellular localization of ELF3 in my experimental samples?

ELF3 exhibits both nuclear and cytoplasmic localization, which carries important biological implications:

Interpreting localization patterns:

• Nuclear localization typically indicates active transcriptional regulation

• Cytoplasmic localization may represent alternative functions or regulatory mechanisms

• Changes in localization pattern may correlate with disease progression or cellular state

Immunohistochemistry and immunofluorescence studies have demonstrated both nuclear and cytoplasmic ELF3 localization in human tumors and cell lines . This dual localization pattern suggests that ELF3 may have as-yet uncharacterized functions beyond its recognized role as a transcription factor.

When analyzing localization data, researchers should:

• Quantify nuclear-to-cytoplasmic ratios across experimental conditions

• Correlate localization patterns with functional outcomes (proliferation, migration, etc.)

• Investigate mechanisms controlling ELF3 trafficking between compartments

• Consider phosphorylation status or other post-translational modifications that might affect localization

Interestingly, ELF3 has been reported to transform breast cells through a cytoplasmic mechanism, highlighting the functional significance of its subcellular distribution .

What approaches can help resolve contradictory findings regarding ELF3's function across different cancer models?

The seemingly contradictory roles of ELF3 across cancer types necessitate careful experimental design:

Strategies to address contradictory findings:

• Conduct parallel experiments in multiple cell lines representing different tissue origins

• Control for genetic background by using isogenic cell systems with defined genetic alterations

• Employ in vivo models that better recapitulate tissue microenvironment

• Classify cancer subtypes more precisely using molecular profiling before assessing ELF3 function

Research reveals that ELF3 displays oncogenic properties in lung adenocarcinoma but tumor-suppressive functions in other epithelial cancers . Molecular mechanisms underlying this duality may include:

• Tissue-specific co-factor availability

• Differential chromatin landscapes affecting target gene accessibility

• Varying post-translational modifications

• Distinct protein-protein interaction networks across tissue types

The molecular context is crucial - ELF3 disruption occurs across multiple molecular subtypes of lung adenocarcinoma, with high expression occurring regardless of KRAS or EGFR mutation status . This suggests that ELF3's function may be determined by broader tissue-specific programs rather than specific oncogenic drivers.

How can I design experiments to investigate ELF3's role in epithelial-to-mesenchymal transition?

ELF3's connection to epithelial identity makes it a valuable target for EMT studies:

Key experimental design considerations:

• Monitor ELF3 expression during induced EMT (via TGF-β, hypoxia, or other EMT inducers)

• Correlate ELF3 levels with epithelial markers (E-cadherin) and mesenchymal markers (Vimentin, N-cadherin)

• Assess impact of ELF3 modulation on EMT-promoting transcription factors (ZEB1, SNAIL, TWIST)

• Evaluate functional EMT outcomes (migration, invasion, resistance to anoikis) following ELF3 manipulation

Research has established connections between ELF3 and epithelial identity, with positive associations between ELF3 and E-cadherin (epithelial marker) and negative correlations with EMT-promoting ZEB1 . These relationships can be exploited to understand how ELF3 influences epithelial plasticity and cancer progression.

When designing EMT-focused experiments, consider these approaches:

• Use 3D culture systems or organoids to better recapitulate epithelial architecture

• Employ live-cell imaging to track dynamic changes in cell morphology and migration

• Integrate transcriptomic and proteomic analyses to identify EMT-related targets of ELF3

• Compare results across multiple model systems to distinguish tissue-specific from universal functions