Phospho-ESR1 (Ser104) Antibody

What is Phospho-ESR1 (Ser104) Antibody and what is its significance in research?

Phospho-ESR1 (Ser104) Antibody is a specialized antibody that specifically recognizes the estrogen receptor alpha (ERα) when phosphorylated at serine residue 104. This antibody is typically produced by immunizing rabbits with synthetic phosphopeptides containing the phosphorylated S104 sequence conjugated to KLH (keyhole limpet hemocyanin) . The significance of this antibody lies in its ability to detect post-translational modifications that regulate ERα activity, which is crucial for understanding estrogen signaling pathways in normal physiology and disease states.

ERα phosphorylation at specific residues in transcription activation function 1 (AF-1), including serine 104, has been shown to stimulate receptor activity in a ligand-independent manner . This phosphorylation event is particularly important in breast cancer research as it may contribute to mechanisms of endocrine therapy resistance, making phospho-specific antibodies valuable tools for investigating altered estrogen signaling in cancer progression .

What experimental applications are most suitable for Phospho-ESR1 (Ser104) Antibody?

Phospho-ESR1 (Ser104) Antibody has demonstrated utility in multiple experimental applications:

• Western Blot (WB): Typically used at dilutions of 1:500-1:1000 for detecting phosphorylated ERα in cell or tissue lysates .

• Immunohistochemistry (IHC): Effective at dilutions of 1:50-1:100 for examining phospho-ERα localization and expression in tissue sections .

• ELISA: Particularly useful in cell-based ELISA formats for quantifying relative amounts of phosphorylated ERα in cultured cells .

The antibody shows reactivity with human and mouse samples, making it suitable for comparative studies across these species . For optimal results, experimental conditions should be validated and optimized for each specific application and sample type.

How can phosphorylation at Ser104 be induced or manipulated in experimental settings?

Phosphorylation of ERα at Ser104 can be experimentally induced or manipulated through several approaches:

• MAPK pathway activation: Treatment with PMA (phorbol 12-myristate 13-acetate) activates the ERK1/2 MAPK pathway, leading to increased phosphorylation at Ser104 .

• Estrogen treatment: Exposure to estradiol (E2) stimulates phosphorylation at Ser104, though this may involve both ligand-dependent and MAPK-mediated mechanisms .

• Raf/Ras activation: Expression of constitutively active Raf or Ras can induce Ser104 phosphorylation through MAPK pathway stimulation .

• Kinase inhibition studies: The MEK1/2 inhibitor U0126 can block phosphorylation at Ser104, confirming the role of the MAPK pathway and providing a negative control for experimental validation .

These experimental manipulations allow researchers to study the functional consequences of Ser104 phosphorylation in various cellular contexts.

How does phosphorylation at Ser104 interact with other ERα phosphorylation sites?

Phosphorylation at Ser104 demonstrates complex interrelationships with other ERα phosphorylation sites, particularly Ser106 and Ser118, which are also located within the AF-1 domain. Research indicates a hierarchical and potentially cooperative relationship between these sites:

• Interdependence with Ser106 and Ser118: Phosphorylation status at Ser104 is influenced by the phosphorylation state of Ser106 and Ser118 . When Ser106 or Ser118 are substituted with alanine (preventing phosphorylation), Ser104 phosphorylation is reduced, suggesting that prior phosphorylation at these sites may facilitate Ser104 phosphorylation .

• Sequential phosphorylation: Evidence suggests that phosphorylation at Ser118 may precede and enhance phosphorylation at Ser106, which in turn may promote phosphorylation at Ser104 . This sequential pattern indicates a coordinated regulation of multiple phosphorylation events.

• Functional synergy: Combined phosphorylation at Ser104, Ser106, and Ser118 appears to produce synergistic effects on ERα transcriptional activity that exceed the effects of phosphorylation at individual sites .

These interactions highlight the complexity of ERα regulation through multisite phosphorylation and emphasize the importance of considering the phosphorylation status of all three sites when studying ERα function.

What role does Ser104 phosphorylation play in tamoxifen resistance mechanisms?

Phosphorylation of ERα at Ser104 contributes to tamoxifen resistance through several mechanisms:

• Enhanced AF-1 activity: Phosphorylation at Ser104, along with Ser106, enhances the activity of the AF-1 domain, which can partially compensate for the antagonism of the AF-2 domain by tamoxifen, converting tamoxifen from an antagonist to a partial agonist .

• 4-hydroxytamoxifen (OHT) agonist activity: Research shows that Ser104 and Ser106 are required for the agonist activity of OHT, alongside the well-established role of Ser118 . Mutation of these sites to alanine reduces the ability of OHT to stimulate ERα transcriptional activity.

• MAPK hyperactivation: In some breast cancer cells, hyperactivation of MAPK signaling leads to increased phosphorylation at Ser104, Ser106, and Ser118, potentially contributing to tamoxifen resistance through ligand-independent activation of ERα .

• Altered coregulator recruitment: Phosphorylation at these sites may modify interactions with transcriptional coregulators, shifting the balance from corepressor to coactivator binding in the presence of tamoxifen.

Understanding these mechanisms is crucial for developing strategies to overcome or prevent tamoxifen resistance in hormone-dependent breast cancers.

What methodological considerations are important when detecting Phospho-ERα (Ser104) in complex samples?

Detecting Phospho-ERα (Ser104) in complex biological samples requires careful methodological consideration:

• Antibody validation: Confirm antibody specificity using appropriate controls:

  • Phosphopeptide competition assays to verify phospho-specificity

  • Comparison of wild-type ERα with S104A mutants

  • Inclusion of phosphatase-treated samples as negative controls

• Sample preparation: Phosphorylation status can be labile:

  • Use phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in all buffers

  • Process samples rapidly and maintain cold temperatures

  • Consider crosslinking agents for IHC applications to preserve phosphoepitopes

• Signal normalization strategies:

  • Use total ERα antibodies for normalization to account for variations in total protein expression

  • Include GAPDH as an internal loading control

  • For cell-based assays, normalize to cell number using Crystal Violet staining

• Considerations for specific techniques:

  • For Western blotting: Use 7-8% gels for optimal resolution of phosphorylated forms

  • For IHC: Optimize antigen retrieval conditions (pH, temperature, duration)

  • For ELISA: Be aware of potential cross-reactivity with other phosphorylation sites

These methodological considerations help ensure reliable and reproducible detection of Phospho-ERα (Ser104) in experimental settings.

How can I design experiments to investigate the functional consequences of Ser104 phosphorylation?

Designing experiments to investigate the functional significance of Ser104 phosphorylation requires multiple complementary approaches:

• Site-directed mutagenesis strategies:

  • Generate S104A mutants (preventing phosphorylation) to study loss-of-function effects

  • Create S104E or S104D phosphomimetic mutants to model constitutive phosphorylation

  • Develop multiple mutants (e.g., S104A/S106A/S118A) to study combinatorial effects

• Reporter gene assays:

  • Transfect cells with ERE-luciferase reporters alongside wild-type or mutant ERα

  • Compare transcriptional activity in response to different ligands (estradiol, SERMs)

  • Examine how MAPK pathway modulators affect wild-type versus mutant ERα activity

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with wild-type and mutant ERα

  • Use mammalian two-hybrid assays to identify differential coregulator recruitment

  • Apply proximity ligation assays to visualize interactions in situ

• Chromatin immunoprecipitation (ChIP):

  • Compare binding of wild-type and phospho-mutant ERα to target gene promoters

  • Investigate recruitment of coregulators in association with phosphorylated ERα

  • Perform sequential ChIP to examine multiple modifications simultaneously

These experimental approaches provide complementary data about how Ser104 phosphorylation affects ERα function at molecular, cellular, and genomic levels.

What are the common challenges and solutions when working with phospho-specific antibodies like Phospho-ESR1 (Ser104)?

Working with phospho-specific antibodies presents several challenges that require specific solutions:

Additional strategies to improve phospho-specific detection include using signal amplification techniques like tyramide signal amplification for IHC, or deploying proximity ligation assays to detect specific protein-protein interactions only when phosphorylation is present.

How should quantitative analysis of Phospho-ESR1 (Ser104) be performed and interpreted?

Quantitative analysis of Phospho-ESR1 (Ser104) requires rigorous methodology and careful interpretation:

• Normalization strategies:

  • Always normalize phospho-signal to total ERα expression to account for variations in expression levels

  • Use housekeeping proteins (GAPDH) as loading controls for Western blot and cell-based assays

  • For cell-based assays, normalize to cell number using Crystal Violet staining to adjust for plating differences

• Quantification methods:

  • For Western blots: Use digital image analysis with linear dynamic range verification

  • For IHC: Apply H-score or Allred scoring systems with blinded evaluation

  • For ELISA: Generate standard curves using recombinant proteins when possible

• Statistical analysis:

  • Compare phospho/total ratios rather than absolute phospho-signal

  • Use appropriate statistical tests based on data distribution

  • Consider biological replicates (different samples) separately from technical replicates

• Interpretation considerations:

  • Context-dependent phosphorylation may vary by cell type, stimulus, and time point

  • Correlation with functional outcomes requires parallel assays (transcription, proliferation)

  • Phosphorylation at S104 should be interpreted in conjunction with S106 and S118 status due to their interdependence

• Validation approaches:

  • Confirm key findings with alternative detection methods

  • Use phosphatase treatment as negative control

  • Employ MAPK inhibitors (U0126) to demonstrate specificity of the phosphorylation mechanism

Following these guidelines ensures robust quantitative analysis of Phospho-ESR1 (Ser104) and meaningful interpretation of experimental results.

How do cell-based ELISA techniques compare with other methods for studying Phospho-ESR1 (Ser104)?

Cell-based ELISA techniques offer distinct advantages and limitations compared to other methods for studying Phospho-ESR1 (Ser104):

Cell-based ELISA techniques are particularly valuable for high-throughput screening of compounds or conditions that affect ERα phosphorylation at Ser104, making them ideal for initial screens that can later be validated with more detailed but lower-throughput methods .

What emerging techniques might enhance our understanding of ERα Ser104 phosphorylation dynamics?

Several emerging technologies hold promise for advancing our understanding of ERα Ser104 phosphorylation dynamics:

• Phospho-specific proximity ligation assays (PLA):

  • Enables visualization of phosphorylated ERα interactions with specific cofactors

  • Provides single-molecule sensitivity in intact cells

  • Can reveal cell-to-cell variability in phosphorylation status

• CRISPR-Cas9 genome editing:

  • Generation of endogenous S104A or S104E knock-in cell lines

  • Creates physiologically relevant models with normal expression levels

  • Eliminates artifacts associated with overexpression systems

• Phospho-proteomic approaches:

  • Tandem mass tag (TMT) labeling for quantitative comparisons across conditions

  • Phospho-enrichment strategies to enhance detection sensitivity

  • Integration with other -omics data for systems-level insights

• Live-cell phosphorylation sensors:

  • FRET-based biosensors to monitor Ser104 phosphorylation in real-time

  • Optogenetic approaches to control kinase activity with spatial precision

  • Single-cell tracking of phosphorylation dynamics

• Spatial transcriptomics:

  • Correlating Ser104 phosphorylation with transcriptional outputs in tissue context

  • Understanding heterogeneity of phosphorylation and its consequences

  • Linking phosphorylation to microenvironmental factors

These emerging techniques will provide unprecedented temporal and spatial resolution for studying ERα phosphorylation dynamics, potentially revealing new aspects of estrogen signaling in normal physiology and disease states.

How might Phospho-ESR1 (Ser104) research contribute to precision medicine approaches for breast cancer?

Research on Phospho-ESR1 (Ser104) has several potential applications in precision medicine for breast cancer:

• Biomarker development:

  • Phospho-S104 levels could serve as predictive biomarkers for response to endocrine therapies

  • The ratio of phosphorylated to total ERα might identify patients at risk for resistance

  • Combined assessment of S104, S106, and S118 phosphorylation could provide a "phospho-signature" with greater predictive value than single markers

• Therapeutic target identification:

  • Understanding the kinases responsible for S104 phosphorylation (ERK1/2 MAPK) supports the rational combination of MAPK inhibitors with endocrine therapies

  • Agents that specifically block the consequences of S104 phosphorylation without affecting beneficial ERα functions could minimize side effects

• Resistance mechanism characterization:

  • Phosphorylation at S104 appears to contribute to tamoxifen resistance by enhancing the agonist properties of 4-hydroxytamoxifen

  • This insight can guide therapy selection, suggesting that patients with elevated S104 phosphorylation might benefit from alternative therapies

• Patient stratification strategies:

  • Classifying tumors based on ERα phosphorylation patterns could identify distinct biological subgroups within ERα-positive breast cancers

  • This stratification could inform clinical trial design and interpretation, potentially explaining differential responses to targeted therapies

• Monitoring treatment response:

  • Serial assessment of phosphorylation status could provide early indicators of developing resistance

  • Dynamic changes in phosphorylation might guide adaptive therapy approaches

These applications highlight how detailed understanding of ERα phosphorylation at S104 could translate into clinically relevant tools for personalizing breast cancer treatment and improving patient outcomes.

How should researchers interpret conflicting results in Phospho-ESR1 (Ser104) detection across different experimental systems?

When faced with conflicting results in Phospho-ESR1 (Ser104) detection across different experimental systems, researchers should consider several factors:

• Antibody variables:

  • Different antibodies may have varying specificity and sensitivity for the phospho-epitope

  • Polyclonal antibodies can show batch-to-batch variation

  • Confirm results with multiple antibodies or alternative detection methods

• Cell/tissue context considerations:

  • Phosphorylation patterns may legitimately differ between cell types due to different kinase activities

  • Primary tissues versus cell lines may show different baseline phosphorylation levels

  • Microenvironmental factors can influence phosphorylation status

• Technical variables to evaluate:

  • Sample preparation methods (lysis buffers, fixation protocols)

  • Presence and concentration of phosphatase inhibitors

  • Time between sample collection and analysis

  • Detection method sensitivity thresholds

• Biological complexity:

  • Interdependence between phosphorylation sites may affect epitope availability

  • Temporal dynamics of phosphorylation (rapid vs. sustained)

  • Heterogeneity within samples (single-cell methods may reveal subpopulations)

• Validation approaches:

  • Use phosphatase treatment as a negative control

  • Include S104A mutants as specificity controls

  • Apply MAPK pathway inhibitors (U0126) to confirm pathway-specific phosphorylation

  • Consider phosphopeptide competition assays to verify antibody specificity

By systematically addressing these factors, researchers can resolve apparent contradictions and develop a more nuanced understanding of the biological variability in ERα phosphorylation.

What are the most appropriate positive and negative controls for Phospho-ESR1 (Ser104) experiments?

Establishing appropriate controls is critical for robust Phospho-ESR1 (Ser104) experiments:

Positive Controls:

• Stimulated cell lysates:

  • Cells treated with PMA to activate MAPK signaling

  • Estradiol (E2)-treated cells expressing wild-type ERα

  • Cells expressing constitutively active Raf or Ras

• Phosphomimetic mutants:

  • S104E or S104D ERα mutants that mimic phosphorylation

  • Can serve as controls for functional studies, though they may not perfectly replicate phosphorylation effects

• Known positive samples:

  • MCF-7 breast cancer cells treated with growth factors

  • Certain breast cancer tissue samples with known MAPK activation

Negative Controls:

• Phosphorylation site mutants:

  • S104A mutant ERα (prevents phosphorylation at the specific site)

  • Allows discrimination between specific and non-specific antibody binding

• Phosphatase-treated samples:

  • Treatment with lambda phosphatase to remove phosphate groups

  • Verifies that signal depends on phosphorylation status

• Kinase inhibition:

  • U0126 (MEK1/2 inhibitor) treatment to block MAPK-mediated phosphorylation

  • Provides pathway-specific negative control

• Peptide competition:

  • Pre-incubation of antibody with phosphorylated peptide containing the S104 epitope

  • Should abolish specific signal while non-phosphorylated peptide should have minimal effect

• ERα-negative cells:

  • Cell lines lacking ERα expression

  • Controls for non-specific antibody binding

Using these positive and negative controls systematically enhances the reliability and interpretability of Phospho-ESR1 (Ser104) experiments.

How can phosphorylation at Ser104 be distinguished from other post-translational modifications of ERα?

Distinguishing phosphorylation at Ser104 from other post-translational modifications (PTMs) of ERα requires specific experimental approaches:

• Antibody-based methods with enhanced specificity:

  • Use antibodies that recognize specific phosphorylated residues and their surrounding sequence context

  • Perform peptide competition assays with phosphorylated and non-phosphorylated peptides to confirm specificity

  • Apply multiple antibodies targeting different epitopes containing the same modification

• Mutational analyses:

  • Generate site-specific mutants (S104A) to prevent phosphorylation at the site of interest

  • Compare with wild-type ERα and other PTM site mutants

  • Create combinatorial mutants to study interactions between modifications

• Mass spectrometry approaches:

  • Perform tandem MS (MS/MS) to identify specific modified residues

  • Use neutral loss scanning to detect phosphorylation

  • Apply multiple fragmentation methods (CID, ETD, HCD) for comprehensive PTM mapping

  • Quantify modification stoichiometry at specific sites

• Enzymatic treatments:

  • Use phosphatase treatment to remove phosphorylation

  • Apply deubiquitinases or deacetylases to remove other specific PTMs

  • Compare modification patterns before and after treatment

• Temporal dynamics and stimulus specificity:

  • Different PTMs may show distinct temporal patterns after stimulation

  • MAPK activators (PMA) specifically enhance Ser104 phosphorylation

  • Compare responses to different stimuli to distinguish pathway-specific modifications

By combining these approaches, researchers can confidently distinguish phosphorylation at Ser104 from other PTMs and understand their potentially independent or cooperative effects on ERα function.