ESR1 Antibody

What is ESR1 and why is it important in research?

ESR1, also known as estrogen receptor alpha (ERα), is a member of the steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors. It plays a critical role in regulating gene expression, development and differentiation (particularly in female reproductive tissues), reproduction and fertility, metabolism, cardiovascular health, and cancer development. ESR1 is approximately 66.2 kDa in size and has multiple isoforms . It binds to estrogen and regulates the expression of genes involved in cell proliferation, differentiation, and apoptosis, making it a significant target in oncology research, particularly for hormone-receptor positive breast cancers .

What types of ESR1 antibodies are available for research applications?

ESR1 antibodies are available in multiple formats:

• Monoclonal antibodies: Provide high specificity and consistency between batches; examples include recombinant monoclonal antibodies like CSB-RA172909A0HU

• Polyclonal antibodies: Recognize multiple epitopes, potentially providing stronger signals; examples include rabbit polyclonal antibodies like A00057

• Application-specific antibodies: Optimized for particular techniques such as Western blot, IHC, IF, or ELISA

The selection depends on the specific research application, required specificity, and target species reactivity.

What is the molecular structure and domain organization of ESR1?

ESR1 contains several functional domains critical for its activity:

• DNA binding domain that interacts with estrogen response elements

• Ligand binding domain that recognizes estrogen

• N-terminal transcription activation function-1 domain (AF-1), where phosphorylation of serines 104 and 106 regulates ESR1 activity

The protein has a predicted amino acid length of 595 and a mass of 66.2 kDa. Currently, there are 4 reported isoforms . The immunogen for many commercially available antibodies typically targets specific regions, such as an 18 amino acid peptide near the center of human ESR1 or the region within amino acids 250-300 .

What are the recommended protocols for using ESR1 antibodies in Western blot applications?

When using ESR1 antibodies for Western blot:

• Sample preparation:

  • Use fresh tissue or cell lysates

  • Include appropriate positive controls (e.g., MCF-7 cell lysate for human samples)

• Recommended dilutions:

  • Typical working dilutions range from 1:500 to 1:5000 depending on the antibody

  • For A00057, the recommended dilution is 1-2 μg/mL

• Expected molecular weight:

  • The observed molecular weight is approximately 68 kDa

  • The calculated molecular weight is 66.2 kDa

• Validation controls:

  • Include blocking peptides to confirm specificity

  • Use appropriate negative controls like ESR1-negative cell lines

For optimal results, antibody dilutions should be empirically determined for each specific application and sample type.

How should ESR1 antibodies be applied in immunohistochemistry (IHC) techniques?

For successful IHC applications with ESR1 antibodies:

• Tissue preparation:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissue sections

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective

• Protocol optimization:

  • For antibody A00057, use at approximately 5 μg/mL concentration

  • For monoclonal antibodies like those from Abbexa, use dilutions between 1:200-1:1000

• Signal detection:

  • For nuclear proteins like ESR1, use a high-contrast detection system

  • Include positive controls such as breast cancer tissue known to express ESR1

• Counterstaining and interpretation:

  • Hematoxylin counterstain provides nuclear contrast

  • Evaluate nuclear localization of ESR1 staining

  • Consider both intensity and percentage of positive cells for scoring

Careful optimization of antigen retrieval conditions is critical for consistent results with nuclear receptors like ESR1.

What methodological approach is recommended for detecting ESR1 mutations in patient samples?

Based on recent research methodologies, a multi-step approach for detecting ESR1 mutations is recommended:

• CTC enrichment: Using technologies like CellSearch® for initial capture of circulating tumor cells from blood samples

• Single cell isolation: Employing DEPArray™ technology to isolate individual CTCs for molecular analysis

• Whole genome amplification: Applying methods like MALBAC (Multiple Annealing and Looping-Based Amplification Cycles) to amplify the genetic material from single cells

• Mutation detection: Performing Sanger sequencing to identify mutations in the ESR1 gene

This approach allows for detecting ESR1 mutations at the single circulating tumor cell level in patients with metastatic breast cancer, which is particularly valuable for monitoring resistance to endocrine therapy and guiding treatment decisions .

How can researchers assess antibody specificity and validate ESR1 antibodies for their experiments?

Comprehensive validation of ESR1 antibodies should include:

• Cross-reactivity testing:

  • Verify species reactivity (human, mouse, rat, etc.) as claimed by the manufacturer

  • Test antibody in known positive and negative tissue/cell samples

• Specificity validation:

  • Perform blocking peptide experiments where the immunizing peptide blocks antibody binding

  • Compare staining patterns with multiple antibodies targeting different epitopes

  • Use genetic knockdown/knockout samples as negative controls

• Application-specific validation:

  • For each application (WB, IHC, IF, ELISA), perform separate validation experiments

  • Document antibody performance metrics including sensitivity and signal-to-noise ratio

• Reproducibility assessment:

  • Test multiple antibody lots if available

  • Compare with published literature and expected expression patterns

A well-validated antibody should show consistent results across multiple experimental conditions and align with established ESR1 expression patterns in appropriate tissues.

What are the challenges in detecting ESR1 mutations in circulating tumor cells and how can they be overcome?

Detecting ESR1 mutations in circulating tumor cells faces several challenges:

• Rarity of CTCs: CTCs are extremely rare, constituting approximately 1-10 cells per mL of blood in patients with metastatic disease

  • Solution: Use highly sensitive enrichment methods like CellSearch® followed by DEPArray™ for precise single-cell isolation

• Heterogeneity: Significant heterogeneity exists among CTCs from the same patient

  • Solution: Analyze multiple individual CTCs rather than pooled samples to capture the full spectrum of mutations

• Limited genetic material: Single CTCs provide minimal amounts of DNA for analysis

  • Solution: Employ whole genome amplification techniques like MALBAC to generate sufficient material for sequencing

• False positives/negatives: Technical artifacts can arise during amplification of single-cell DNA

  • Solution: Include appropriate controls such as white blood cells from the same patient to establish baseline and validate findings

The integrated workflow combining CellSearch® enrichment, DEPArray™ isolation, MALBAC amplification, and Sanger sequencing has proven effective in overcoming these challenges for reliable ESR1 mutation detection .

How do post-translational modifications of ESR1 affect antibody selection and experimental design?

Post-translational modifications (PTMs) of ESR1 critically influence its function and can affect antibody recognition:

• Common ESR1 PTMs:

  • Phosphorylation, particularly of serines 104 and 106 in the AF-1 domain, regulates ESR1 activity

  • Acetylation, methylation, and SUMOylation can also occur

• Antibody epitope considerations:

  • PTM-specific antibodies recognize only particular modified forms of ESR1

  • Standard antibodies may have reduced binding to heavily modified ESR1

  • The immunogen location is crucial - antibodies targeting regions around amino acids 250-300 may have different sensitivity to PTMs than those targeting other domains

• Experimental design implications:

  • When studying activation states, consider phospho-specific antibodies

  • For total ESR1 detection regardless of activation, choose antibodies with epitopes in regions less affected by PTMs

  • Use phosphatase treatments as controls when studying phosphorylation-dependent phenomena

• Validation strategies:

  • Compare results with multiple antibodies recognizing different epitopes

  • Include appropriate controls that modulate the PTM status (kinase/phosphatase inhibitors)

Understanding the relationship between ESR1 PTMs and antibody recognition is essential for accurate interpretation of experimental data, particularly in signaling studies.

What are common issues with ESR1 antibody staining in IHC and how can they be resolved?

For optimal IHC results with ESR1 antibodies like the validated A00057, researchers should meticulously optimize each step of the protocol, particularly antigen retrieval conditions and antibody concentration (recommended at 5 μg/mL for IHC applications) .

How can researchers optimize Western blot protocols for detecting ESR1 in different tissue samples?

Optimizing Western blot protocols for ESR1 detection requires attention to several key factors:

• Sample preparation optimization:

  • For brain tissue (as validated with A00057): Use gentle lysis buffers containing protease inhibitors to preserve the 68 kDa ESR1 protein

  • For reproductive tissues: Consider using specialized extraction buffers that account for high lipid content

  • Include phosphatase inhibitors if studying phosphorylated ESR1 forms

• Protein loading and transfer parameters:

  • Load 20-50 μg of total protein depending on expected ESR1 expression levels

  • Use optimized transfer conditions for proteins in the 65-70 kDa range

  • Consider semi-dry transfer for 1-1.5 hours or wet transfer overnight at 30V

• Antibody incubation optimization:

  • For A00057: Use 1-2 μg/mL in blocking buffer

  • Incubate primary antibody at 4°C overnight for optimal signal-to-noise ratio

  • For monoclonal antibodies: Test dilution ranges from 1:500-1:2000

• Validation with controls:

  • Include a blocking peptide control to confirm specificity

  • Use known positive control lysates (e.g., MCF-7 cells for human ESR1)

  • Consider including a ladder specific for the 50-100 kDa range for precise sizing

By carefully optimizing these parameters for each tissue type, researchers can achieve consistent and specific detection of ESR1 protein.

How are ESR1 antibodies being utilized in single-cell analysis techniques for cancer research?

ESR1 antibodies are playing an increasingly important role in single-cell analysis for cancer research:

• Circulating tumor cell (CTC) characterization:

  • ESR1 antibodies are used to identify and isolate ESR1-positive CTCs from blood samples of breast cancer patients

  • Combined with DEPArray™ technology, this enables single-cell isolation for downstream molecular analysis

  • This approach allows monitoring of ESR1 mutations that emerge during endocrine therapy resistance

• Integrated multi-omic approaches:

  • ESR1 antibody-based cell sorting can be combined with single-cell RNA sequencing

  • This integration allows correlation of ESR1 protein levels with transcriptomic profiles

  • Researchers can identify distinct cellular subpopulations based on ESR1 expression and activation states

• Spatial analysis in tumor microenvironments:

  • ESR1 antibodies are employed in multiplexed immunofluorescence to map ESR1-expressing cells within complex tumor tissues

  • This spatial information provides insights into tumor heterogeneity and microenvironmental interactions

• Functional single-cell assays:

  • ESR1 antibodies enable sorting of viable cells for downstream functional experiments

  • This facilitates the study of ESR1-positive cell subpopulations and their behaviors

These applications demonstrate how ESR1 antibodies contribute to understanding the heterogeneity of estrogen-responsive cancers at the single-cell level, with important implications for personalized medicine approaches .

What is the role of ESR1 antibodies in studying endocrine resistance mechanisms in breast cancer?

ESR1 antibodies have become essential tools for investigating the complex mechanisms of endocrine resistance in breast cancer:

• Detection of ESR1 mutations:

  • ESR1 antibodies facilitate the isolation of CTCs for analysis of activating ESR1 mutations that confer resistance to endocrine therapy

  • The established workflow combining CellSearch®, DEPArray™, and molecular analysis enables monitoring of these mutations during treatment

• Analysis of ESR1 expression dynamics:

  • Antibodies help track changes in ESR1 expression levels before, during, and after development of resistance

  • This temporal analysis provides insights into adaptive mechanisms employed by cancer cells

• Investigation of altered ESR1 signaling:

  • Phospho-specific ESR1 antibodies detect changes in activation patterns associated with resistance

  • Co-immunoprecipitation studies using ESR1 antibodies reveal altered protein-protein interactions in resistant cells

• Evaluation of ESR1 splice variants:

  • Antibodies targeting different domains can distinguish between full-length ESR1 and truncated variants that may contribute to resistance

  • This approach helps identify the prevalence of particular variants in resistant populations

The systematic application of ESR1 antibodies in these contexts has significantly advanced our understanding of endocrine resistance mechanisms, potentially leading to novel therapeutic strategies for hormone receptor-positive breast cancers that develop resistance to standard treatments .

How can researchers effectively combine ESR1 antibodies with other molecular techniques for comprehensive pathway analysis?

Integrating ESR1 antibodies with complementary molecular techniques enables comprehensive pathway analysis:

• ChIP-seq integration:

  • ESR1 antibodies can be used for chromatin immunoprecipitation followed by sequencing (ChIP-seq)

  • This identifies genome-wide ESR1 binding sites and can be correlated with RNA-seq data to link binding events with transcriptional outcomes

  • For optimal results, use ChIP-validated antibodies with high specificity for ESR1

• Proximity ligation assays (PLA):

  • Combining ESR1 antibodies with antibodies against potential interacting proteins in PLA

  • This approach visualizes and quantifies protein-protein interactions in situ

  • Particularly valuable for studying ESR1 interactions with coactivators, corepressors, and other transcription factors

• Mass spectrometry-based proteomics:

  • ESR1 antibodies enable immunoprecipitation of ESR1 complexes for mass spectrometry analysis

  • This identifies novel binding partners and post-translational modifications

  • Integration with phosphoproteomics reveals signaling cascades downstream of ESR1 activation

• Spatial transcriptomics coordination:

  • ESR1 antibody-based immunostaining can be aligned with spatial transcriptomics data

  • This correlation links ESR1 protein expression with local gene expression profiles

  • Provides insights into the spatial organization of estrogen-responsive gene programs

By strategically combining these approaches, researchers can build comprehensive models of ESR1 signaling networks, capturing both protein-level interactions and transcriptional consequences in various physiological and pathological contexts.

What are the emerging technologies for detecting ESR1 mutations that might complement antibody-based approaches?

Several emerging technologies show promise for ESR1 mutation detection that could complement antibody-based methods:

• Digital PCR technologies:

  • Droplet digital PCR (ddPCR) offers ultrasensitive detection of ESR1 mutations

  • Can detect mutations at frequencies as low as 0.1%, outperforming traditional sequencing

  • Particularly valuable for liquid biopsy samples with low mutation abundance

• Next-generation sequencing advances:

  • Targeted panel sequencing with molecular barcoding improves sensitivity for ESR1 mutations

  • Single-cell sequencing technologies can directly analyze ESR1 mutations in individual CTCs

  • These approaches could complement the antibody-based CTC isolation described in the research

• Nanopore sequencing:

  • Long-read sequencing technologies may better detect structural variants in ESR1

  • Direct DNA sequencing without amplification reduces PCR-related artifacts

  • Potential for point-of-care applications with portable devices

• CRISPR-based diagnostic systems:

  • CRISPR-Cas12/13-based detection methods could provide rapid, sensitive detection of known ESR1 mutations

  • These systems might offer point-of-care testing options for monitoring resistance-associated mutations

The integration of these molecular technologies with antibody-based CTC enrichment approaches will likely enhance both sensitivity and specificity for ESR1 mutation detection in clinical samples, ultimately improving patient monitoring and treatment decision-making.

How might advances in recombinant antibody technology impact future ESR1 research?

Advances in recombinant antibody technology are poised to significantly enhance ESR1 research:

• Improved specificity and consistency:

  • Recombinant monoclonal antibodies like those described in the search results offer superior batch-to-batch consistency

  • Gene-sequenced antibodies ensure reproducible epitope targeting

  • This addresses a major challenge in research reproducibility

• Fragment-based antibody derivatives:

  • Single-chain variable fragments (scFvs) and nanobodies against ESR1

  • Smaller size enables better tissue penetration for imaging applications

  • Potential for intracellular expression to monitor ESR1 in living cells

• Multispecific antibodies:

  • Bispecific antibodies targeting ESR1 and other relevant markers simultaneously

  • Enables more sophisticated isolation of specific cell subpopulations

  • Potential for therapeutic applications combining targeting and immune recruitment

• Site-specific conjugation technologies:

  • Precisely engineered antibody-fluorophore or antibody-drug conjugates

  • Improved signal-to-noise ratio for detection applications

  • Maintained antibody functionality after conjugation

These technological advances are likely to provide researchers with more precise tools for ESR1 detection, potentially enabling new applications in both basic research and clinical diagnostics that were previously unattainable with conventional antibody technologies .

What are the implications of ESR1 research for developing novel therapeutic approaches in hormone-dependent cancers?

ESR1 research has significant implications for novel therapeutic strategies:

• Mutation-targeted therapies:

  • Detection of ESR1 mutations using antibody-isolated CTCs helps identify patients who might benefit from novel selective estrogen receptor degraders (SERDs) or modulators

  • This personalized approach may overcome resistance to conventional endocrine therapies

• Combination therapy approaches:

  • Understanding ESR1 signaling pathways through antibody-based research identifies rational combination targets

  • CDK4/6 inhibitors, PI3K/mTOR inhibitors, and HDAC inhibitors have shown promise in combination with endocrine therapy

• Antibody-drug conjugates (ADCs):

  • ESR1 antibodies could potentially be developed into ADCs for targeted delivery of cytotoxic agents to ESR1-positive cells

  • This approach might address heterogeneous ESR1 expression in metastatic disease

• Immune-based approaches:

  • ESR1 peptide vaccines targeting mutated regions could generate immune responses against resistant tumor cells

  • Checkpoint inhibitors combined with endocrine therapy might overcome immune suppression in the tumor microenvironment

• Diagnostic and therapeutic monitoring:

  • The CTC isolation and molecular characterization approach detailed in the research enables real-time monitoring of treatment response and emergence of resistance

  • This facilitates early intervention and therapy adjustment

The continued development of sensitive and specific ESR1 antibodies and their application in cutting-edge research methodologies will be instrumental in advancing these therapeutic strategies toward clinical implementation, potentially improving outcomes for patients with hormone-dependent cancers.