ESR1 Antibody

What is ESR1 and why is it significant for research applications?

ESR1 (Estrogen Receptor 1) is a nuclear receptor protein that functions primarily in chromatin remodeling and transcriptional regulation. It has a canonical amino acid length of 595 residues and a protein mass of 66.2 kilodaltons, with 4 distinct isoforms identified to date. The protein is localized in multiple cellular compartments including the cell membrane, nucleus, and cytoplasm .

ESR1 is particularly significant for research due to its central role in hormone-responsive cancers, especially breast cancer, where mutations in this gene are frequently associated with endocrine therapy resistance. The protein is notably expressed in reproductive tissues including the vagina, fallopian tube, endometrium, cervix, and breast tissue, making it a critical target for reproductive and oncological research .

What are the primary research applications for ESR1 antibodies?

ESR1 antibodies are employed across multiple experimental platforms with Western Blot being the most common application. Other significant applications include:

• Immunohistochemistry (IHC) for tissue expression analysis

• Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative protein detection

• Immunocytochemistry (ICC) for cellular localization studies

• Immunofluorescence (IF) for subcellular visualization

• Chromatin Immunoprecipitation (ChIP) for studying DNA-protein interactions

Each application requires specific consideration of antibody characteristics including specificity, sensitivity, and validated epitope recognition to ensure reliable experimental outcomes in different research contexts.

How should researchers select appropriate ESR1 antibodies for specific experimental applications?

Selection of appropriate ESR1 antibodies should be guided by several technical considerations:

• Epitope specificity: For mutation studies, select antibodies targeting regions outside the ligand-binding domain (LBD) where common mutations (D538G, Y537S) occur to avoid interference with antibody binding

• Isoform recognition: Determine which of the 4 known isoforms need detection and select antibodies accordingly

• Cross-reactivity: Verify cross-reactivity with target species (human, mouse, rat) based on experimental needs

• Validated applications: Confirm the antibody has been validated specifically for your intended application (WB, IHC, IF)

• Monoclonal vs. polyclonal: Consider using monoclonal antibodies for specific epitope recognition and polyclonal for broader detection capacity

The application-specific requirements should guide selection rather than general characteristics or cost considerations.

What protocols deliver optimal results for ESR1 detection in Western Blot applications?

For optimal ESR1 detection via Western Blot, the following methodological considerations are crucial:

• Sample preparation: Nuclear extraction protocols yield better results than whole cell lysates due to ESR1's predominant nuclear localization

• Protein loading: 20-40 μg of protein per lane is typically sufficient for detection

• Gel percentage: 8-10% SDS-PAGE gels provide optimal separation for the 66.2 kDa ESR1 protein

• Transfer conditions: Semi-dry transfer at 15V for 45 minutes using PVDF membranes shows superior results compared to nitrocellulose

• Blocking: 5% BSA in TBST is preferred over milk-based blocking buffers

• Antibody dilution: Primary antibody concentrations between 1:500-1:2000 typically yield optimal signal-to-noise ratios

• Visualization: Both chemiluminescence and fluorescence-based detection systems are effective, with the latter offering better quantification capacity

Careful optimization of these parameters significantly improves detection sensitivity and specificity, particularly for samples with varying ESR1 expression levels.

How can researchers accurately detect and analyze ESR1 mutations in clinical samples?

Detection of ESR1 mutations requires a methodological approach tailored to the specific research question:

• Sample source selection:

  • Solid tissue biopsy: Provides comprehensive genomic analysis but may miss heterogeneity

  • Circulating tumor cells (CTCs): Enables live cell functional studies

  • Cell-free DNA (cfDNA): Least invasive and captures heterogeneity across multiple metastatic sites

• Detection methods:

  • Next-generation sequencing (NGS): Comprehensive but less sensitive

  • Droplet digital PCR (ddPCR): Highest sensitivity (detecting mutations at 0.1% frequency) but targets only known mutations

• Mutation hotspots: Focus detection on the ligand-binding domain, particularly positions D538G and Y537S (most common), Y537N, Y537C, L536H, L536P, L536R, S463P, and E380Q

• Clonal analysis: Consider that ESR1 mutations are polyclonal in 20-70% of patients, requiring methods that can detect multiple concurrent mutations

For research requiring high sensitivity, ddPCR is recommended as the gold standard methodology, particularly when monitoring disease progression or therapeutic response.

What are the optimal approaches for immunohistochemical detection of ESR1 in tissue samples?

Effective immunohistochemical detection of ESR1 requires specific methodological considerations:

• Fixation: 10% neutral buffered formalin for 24-48 hours provides optimal antigen preservation

• Antigen retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95-98°C for 20 minutes is most effective for ESR1

• Blocking: Use of dual blocking with hydrogen peroxide followed by protein block reduces background

• Primary antibody: Overnight incubation at 4°C with validated anti-ESR1 antibody at 1:100-1:200 dilution

• Detection system: Polymer-based detection systems offer superior sensitivity compared to avidin-biotin methods

• Counterstaining: Brief hematoxylin counterstaining (30-60 seconds) preserves nuclear ESR1 signal visibility

• Controls: Include both positive controls (breast or endometrial tissue) and negative controls (antibody diluent only)

For research applications examining ESR1 mutations, parallel IHC and mutation detection provides valuable correlation between protein expression and mutational status.

How do ESR1 mutations affect protein conformation and antibody binding?

ESR1 mutations induce specific conformational changes that affect both function and antibody recognition:

• Structural alterations: Mutations in the ligand-binding domain (LBD), particularly Y537S and D538G, stabilize the active conformation of ESR1 even in the absence of estrogen

• Constitutive activity: These structural changes lead to increased coactivator recruitment and decreased proteolytic degradation

• Antibody binding implications: Antibodies targeting conformational epitopes in the LBD may show reduced binding affinity for mutated ESR1

• Epitope availability: Mutations alter protein-protein interactions that may mask or expose different epitopes, affecting antibody accessibility

• Detection considerations: Antibodies targeting N-terminal regions typically maintain consistent binding regardless of LBD mutations

For researchers studying ESR1 mutations, using antibodies targeting epitopes outside the commonly mutated regions ensures consistent detection across both wild-type and mutant proteins.

What molecular mechanisms drive ESR1 mutation-mediated therapy resistance?

ESR1 mutations confer resistance through multiple molecular mechanisms that should be considered in research designs:

• Constitutive activity: LBD mutations enable ligand-independent activation, bypassing the need for estrogen binding and conferring resistance to aromatase inhibitors

• Altered drug binding: Structural changes reduce the binding affinity of selective estrogen receptor modulators (SERMs) like tamoxifen

• Degradation resistance: Mutant ESR1 shows decreased susceptibility to selective estrogen receptor degraders (SERDs) such as fulvestrant

• Coactivator recruitment: Enhanced binding to coactivators strengthens downstream signaling even under inhibition conditions

• Target gene alterations: ESR1 mutations modify the transcriptional program, activating genes not typically regulated by wild-type ESR1

These mechanisms explain the clinical observation that approximately 50% of endocrine resistance cases are associated with ESR1 mutations, highlighting the importance of incorporating mutation status in experimental designs.

How do ESR1 mutations influence the molecular subtype characteristics in breast cancer?

ESR1 mutations induce significant molecular subtype alterations that can be studied using appropriate antibodies:

• Basal phenotype acquisition: ESR1 mutant cells show significant enrichment of basal subtype markers, particularly six basal cytokeratins (BCKs) that are among the most enriched genes

• Cytokeratin expression changes: Elevated expression of KRT5, KRT6A, KRT6B, KRT14, KRT16, and KRT17 has been consistently observed in ESR1 mutant cells

• Epigenetic reprogramming: The induction of basal cytokeratins occurs through chromatin reorganization rather than direct ER binding

• Progesterone receptor involvement: This process appears to be orchestrated by progesterone receptor-mediated insulated neighborhood formation

• Immune pathway activation: ESR1 mutant tumors show enrichment of immune pathways similar to basal subtype tumors

These findings challenge the traditional view of ESR1 as a stable luminal lineage marker and suggest that mutations can drive partial subtype switching, which has implications for therapeutic response and experimental design.

How can researchers differentiate between wild-type and mutant ESR1 proteins in experimental systems?

Differentiating between wild-type and mutant ESR1 requires specialized approaches:

• Mutation-specific antibodies: While limited, some research groups have developed antibodies specifically recognizing common mutations like Y537S

• Functional assays: Reporter assays measuring ER activity in the presence/absence of estrogen can indirectly identify constitutively active mutants

• Proximity ligation assays: These can detect altered protein-protein interactions characteristic of mutant ESR1

• Pharmacological response profiling: Differential responses to SERMs and SERDs can distinguish wild-type from mutant ESR1

• Molecular techniques: For definitive identification, complement antibody approaches with:

  • Targeted sequencing

  • Allele-specific PCR

  • Digital droplet PCR (ddPCR)

In experimental systems, creating isogenic cell lines with CRISPR-Cas9 genome editing to introduce specific ESR1 mutations provides valuable tools for comparative studies while controlling for genetic background.

What approaches are recommended for studying ESR1 mutant protein interactions and signaling networks?

Investigating ESR1 mutant protein interactions requires specialized methodological approaches:

• Proximity-based methods:

  • BioID or TurboID for identifying proteins in proximity to wild-type versus mutant ESR1

  • FRET/BRET assays for real-time interaction monitoring

  • Proximity ligation assays for visualizing protein interactions in fixed cells/tissues

• IP-MS workflows:

  • RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins) for chromatin-associated complexes

  • Quantitative AP-MS comparing interactome differences between wild-type and mutant ESR1

  • Crosslinking MS to capture transient interactions

• Chromatin interaction studies:

  • ChIP-seq comparing binding sites between wild-type and mutant ESR1

  • CUT&RUN for higher resolution mapping

  • HiChIP for three-dimensional chromatin interaction landscapes

• Systems biology approaches:

  • Phosphoproteomics to identify differential signaling pathway activation

  • RNA-seq for comprehensive transcriptional output comparison

  • ATAC-seq for chromatin accessibility alterations

These multifaceted approaches provide complementary data that can elucidate how specific mutations alter ESR1 function at molecular and cellular levels.

What methodological considerations are important when detecting ESR1 mutations in heterogeneous clinical samples?

Detection of ESR1 mutations in heterogeneous clinical samples requires specialized approaches:

• Sampling considerations:

  • Serial sampling is recommended due to the acquired nature of ESR1 mutations

  • Multi-site sampling improves detection of heterogeneous mutations

  • Liquid biopsies (cfDNA) capture broader tumor heterogeneity than single-site biopsies

• Technical sensitivity requirements:

  • Detection methods should achieve at least 0.1% variant allele frequency sensitivity

  • Digital PCR methods (ddPCR) generally outperform NGS for low-frequency detection

  • Panel-based testing should include all common hotspot mutations (Y537S, D538G, Y537N, Y537C, L536H/P/R, etc.)

• Bioinformatic analysis:

  • Use algorithms optimized for low variant allele frequency detection

  • Apply error-correction methods for sequencing data

  • Consider clonal relationship analyses to track evolutionary patterns

• Validation approaches:

  • Orthogonal testing using different methodologies

  • Functional confirmation through in vitro models

  • Correlation with treatment outcomes for clinical relevance

These methodological considerations are particularly important as ESR1 mutations often occur as subclonal events with frequencies as low as 1-5% in early resistance stages.

Why might researchers observe variable ESR1 detection in sequential samples and how can this be addressed?

Variability in ESR1 detection across sequential samples can stem from multiple factors that require specific methodological considerations:

• Biological factors:

  • Treatment-induced changes in ESR1 expression

  • Clonal evolution leading to ESR1 mutation emergence or loss

  • Tumor heterogeneity with variable ESR1 expression across sites

• Technical factors:

  • Inconsistent sample processing affecting epitope preservation

  • Antibody lot-to-lot variability impacting detection sensitivity

  • Storage conditions altering protein integrity over time

• Methodological solutions:

  • Standardize preanalytical variables (fixation time, processing protocols)

  • Use calibration controls in each experimental batch

  • Implement dual-antibody approaches targeting different ESR1 epitopes

  • Complement protein detection with mRNA analysis

  • Consider digital pathology quantification for objective assessment

Importantly, some ESR1 mutant cell populations can be lost during continual passage, as demonstrated by the variability in basal cytokeratin detection between early (P6-8) and late passage (P30-32) cell lines . This suggests selection pressures may eliminate certain ESR1 mutant subclones, requiring careful monitoring of model systems across passages.

What factors affect the sensitivity of ESR1 mutation detection in liquid biopsies?

Multiple factors influence ESR1 mutation detection sensitivity in liquid biopsies:

• Preanalytical variables:

  • Blood collection tube type (streck tubes preferred over EDTA)

  • Time between collection and processing (ideally <2 hours)

  • Centrifugation protocol affecting cfDNA yield and quality

  • Storage temperature during transport (-80°C optimal for long-term)

• Technical considerations:

  • Extraction methodology (silica-based methods typically yield higher quality)

  • DNA input amount (minimum 10 ng for reliable detection)

  • Amplicon size (shorter amplicons improve detection in fragmented cfDNA)

  • UMI incorporation for error correction

• Disease-specific factors:

  • Tumor burden correlates with mutation detection sensitivity

  • Metastatic distribution affects cfDNA shedding (liver metastases typically shed more cfDNA)

  • Prior treatments may affect the prevalence of mutations

  • Concurrent genomic alterations may impact detection

Optimizing these variables is critical for reliable detection, particularly when monitoring therapeutic response or minimal residual disease where mutation frequencies may be extremely low.

How can researchers effectively study the functional consequences of ESR1 mutations in experimental systems?

Studying functional consequences of ESR1 mutations requires carefully designed experimental approaches:

• Model system selection:

  • Isogenic cell line pairs (CRISPR-engineered mutations in the same genetic background)

  • Patient-derived models (PDX, organoids, CTCs from ESR1-mutant patients)

  • Transgenic models with inducible ESR1 mutations

• Experimental design considerations:

  • Include multiple ESR1 mutation variants (Y537S, D538G) in parallel

  • Test both estrogen-deprived and estrogen-rich conditions

  • Compare responses across multiple endocrine therapies (AIs, SERMs, SERDs)

  • Evaluate combinations with targeted agents (CDK4/6i, PI3Ki, mTORi)

• Readout selection:

  • Transcriptional reporter assays for ER activity

  • Proliferation in 2D and 3D culture systems

  • Gene expression profiling (RNA-seq, targeted panels)

  • Chromatin accessibility and binding profiles

  • Protein-protein interaction networks

These approaches should be integrated to provide a comprehensive understanding of how specific ESR1 mutations alter signaling, therapeutic response, and cell phenotype.

Emerging Research Directions

Recent research has revealed unexpected immunological consequences of ESR1 mutations:

• Immune pathway activation:

  • ESR1 mutant tumors show enrichment of immune signaling pathways

  • S100A8 and S100A9 are among the most induced immune mediators in ESR1 mutant cells

  • These changes create potential immune therapeutic vulnerabilities

• Tumor microenvironment alterations:

  • Single-cell RNA-seq data indicates ESR1 mutations influence tumor-stroma crosstalk

  • Increased basal cytokeratin expression correlates with altered immune context

  • These changes may enhance immunogenicity of otherwise "cold" luminal tumors

• Research implications:

  • Experimental designs should incorporate immune components when studying ESR1 mutations

  • Combined analysis of ESR1 mutation status and immune infiltration may reveal novel therapeutic targets

  • Preclinical models should maintain intact immune systems to capture these interactions

These findings suggest that ESR1 mutations may create opportunities for immunotherapeutic approaches not typically considered for hormone receptor-positive breast cancers, representing an important new research direction.

How can researchers effectively track ESR1 mutation emergence during therapeutic interventions?

Monitoring ESR1 mutation emergence requires longitudinal sampling approaches:

• Recommended sampling strategy:

  • Baseline assessment before therapy initiation

  • Regular monitoring at 2-3 month intervals during treatment

  • Additional sampling at progression or treatment change points

  • Parallel tissue and liquid biopsies when feasible

• Technological approaches:

  • Digital droplet PCR for known hotspot mutations (highest sensitivity)

  • Targeted NGS panels for broader mutation spectrum detection

  • Ultra-deep sequencing with molecular barcoding for subclonal detection

  • Comparison of ctDNA and CTC analyses for comprehensive assessment

• Analytical considerations:

  • Set predefined thresholds for clinical relevance (typically >0.1% VAF)

  • Implement quality control metrics including minimal input DNA requirements

  • Use time-series analysis to distinguish true emergence from sampling variables

  • Correlate with clinical parameters and imaging findings

This systematic approach enables researchers to track the dynamics of ESR1 mutation emergence, providing insights into resistance mechanisms and potentially guiding therapeutic decision-making in clinical research settings.