Phospho-ESR2 (S87) Antibody

What is Phospho-ESR2 (S87) Antibody and what is its relevance in research?

Phospho-ESR2 (S87) Antibody is a highly specific polyclonal antibody raised in rabbits that recognizes the estrogen receptor beta (ESR2) protein exclusively when phosphorylated at serine 87. This antibody is typically generated using synthetic phosphopeptides derived from human ESR2 around the phosphorylation site [HL(p-S)PL] . Phosphorylation at S87 occurs as part of receptor-ligand interactions that trigger a cascade of events, including dissociation from heat shock proteins, receptor dimerization, and phosphorylation .

The research relevance of this antibody stems from its ability to detect a specific post-translational modification that may regulate ESR2 function. This level of specificity enables researchers to investigate signaling pathways, transcriptional regulation mechanisms, and functional outcomes associated with ESR2 phosphorylation in various biological contexts including cancer, neuroscience, and endocrinology.

What are the validated applications for Phospho-ESR2 (S87) Antibody?

Based on the technical data available, Phospho-ESR2 (S87) Antibody has been validated for the following research applications:

Each application requires specific optimization depending on the experimental system, tissue type, and detection method employed. The antibody demonstrates significant utility across multiple experimental platforms, making it versatile for comprehensive studies of ESR2 phosphorylation.

What species reactivity has been established for Phospho-ESR2 (S87) Antibody?

The documented species reactivity for Phospho-ESR2 (S87) Antibody includes:

• Confirmed reactivity: Mouse

• Predicted reactivity based on sequence homology: Human, Rat, Sheep, and Rabbit

How should experiments be designed to study the kinetics of ESR2 phosphorylation at S87?

Studying phosphorylation kinetics requires careful experimental design:

• Time-course analysis:

  • Establish baseline phosphorylation in serum-starved conditions

  • Stimulate cells with estrogen or specific ESR2 ligands

  • Collect samples at multiple timepoints (0, 5, 15, 30, 60 minutes and 2, 6, 24 hours)

  • Process samples with phosphatase inhibitors present at all steps

• Stimulus concentration dependency:

  • Treat cells with increasing concentrations of stimuli

  • Analyze dose-dependent changes in S87 phosphorylation

  • Determine EC50 values for different ligands

• Quantification methodology:

  • Always normalize phospho-ESR2 signal to total ESR2 levels

  • Use densitometry software with appropriate background correction

  • Generate time-course curves to determine rates of phosphorylation/dephosphorylation

This approach allows determination of both the temporal dynamics and the magnitude of the phosphorylation response under different conditions.

What experimental controls are essential when using Phospho-ESR2 (S87) Antibody?

Rigorous controls are critical for reliable interpretation of results:

• Antibody specificity controls:

  • Lambda phosphatase treatment: Samples treated with phosphatase should show reduced or absent signal

  • Blocking peptide competition: Pre-incubation of antibody with phosphopeptide immunogen should abolish specific signal

  • S87A mutant: Cells expressing phospho-deficient S87A mutant should yield negative results

• Biological controls:

  • Positive control: Samples known to express phosphorylated ESR2 (e.g., estrogen-stimulated cells)

  • Negative control: ESR2 knockout/knockdown samples or tissues known to lack ESR2 expression

  • Treatment controls: Kinase inhibitor treatments targeting known S87 kinases

• Technical controls:

  • Primary antibody omission

  • Isotype control antibody

  • Loading controls for normalization (total ESR2 and housekeeping proteins)

Implementation of these controls ensures reliable data interpretation and enhances reproducibility across experiments.

What sample preparation techniques preserve phosphorylation status of ESR2?

Preserving phosphorylation requires specific precautions:

• Cell/tissue lysis protocol:

  • Use ice-cold lysis buffer containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate)

  • Include protease inhibitors to prevent degradation

  • Maintain samples at 4°C throughout processing

  • Process samples rapidly to minimize dephosphorylation

• Buffer composition:

  • Recommended lysis buffer: 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate

  • Add fresh phosphatase inhibitors: 1 mM Na3VO4, 10 mM NaF, 10 mM β-glycerophosphate

  • Include 1 mM EDTA and 1 mM EGTA to chelate ions required for phosphatase activity

• Sample storage:

  • Aliquot lysates to avoid freeze-thaw cycles

  • Store at -80°C for long-term preservation

  • Add 2X SDS sample buffer immediately after thawing for Western blot applications

These measures minimize artificial dephosphorylation and maximize detection of the physiologically relevant phosphorylation state.

How can Phospho-ESR2 (S87) Antibody be used to investigate cross-talk with other signaling pathways?

Investigating signaling cross-talk requires specialized experimental approaches:

• Co-immunoprecipitation studies:

  • Immunoprecipitate phospho-ESR2 using the S87 antibody

  • Probe for interacting signaling molecules by Western blot

  • Alternatively, immunoprecipitate known interactors and probe for phospho-ESR2

• Pathway inhibitor experiments:

  • Treat cells with inhibitors of specific signaling pathways (MAPK, PI3K/Akt, etc.)

  • Assess effects on ESR2 S87 phosphorylation

  • Identify upstream regulators of the phosphorylation event

• Pathway activation experiments:

  • Activate specific signaling pathways using selective agonists

  • Monitor changes in ESR2 S87 phosphorylation status

  • Establish sequential events in the signaling cascade

• Multiplex analysis:

  • Simultaneously detect phospho-ESR2 alongside other phosphorylated signaling proteins

  • Establish temporal relationships between different phosphorylation events

  • Construct signaling networks based on correlative changes

This systematic approach can reveal how ESR2 phosphorylation integrates with broader cellular signaling networks and identify key regulatory nodes.

How does phosphorylation at S87 impact ESR2 function in transcriptional regulation?

Investigating transcriptional impacts requires specialized nuclear-focused approaches:

• Chromatin immunoprecipitation (ChIP):

  • Use Phospho-ESR2 (S87) Antibody for ChIP experiments

  • Compare binding profiles of phosphorylated vs. total ESR2

  • Identify genes specifically regulated by the phosphorylated form

• Reporter gene assays:

  • Compare transcriptional activity of wild-type ESR2 vs. S87A (non-phosphorylatable) mutant

  • Test responsiveness to different ligands and activating signals

  • Evaluate effects on various estrogen response element configurations

• Transcriptomic analysis:

  • Perform RNA-seq after modulating S87 phosphorylation

  • Compare gene expression profiles between conditions

  • Validate key target genes by qRT-PCR and mechanistic studies

• Co-regulator interaction studies:

  • Assess how S87 phosphorylation affects recruitment of transcriptional co-activators or co-repressors

  • Map domains involved in phosphorylation-dependent interactions

  • Identify co-regulator complexes specific to phosphorylated ESR2

These approaches can establish the functional consequences of S87 phosphorylation on ESR2-mediated transcriptional regulation.

What methodological approaches can address tissue-specific differences in ESR2 phosphorylation patterns?

Tissue-specific analysis requires tailored methodological approaches:

• Tissue microarray analysis:

  • Prepare multi-tissue arrays including various ESR2-expressing tissues

  • Perform immunohistochemistry with Phospho-ESR2 (S87) Antibody

  • Quantify staining intensity and distribution patterns across tissues

• Laser capture microdissection:

  • Isolate specific cell populations from heterogeneous tissues

  • Extract proteins while preserving phosphorylation status

  • Analyze phospho-ESR2 levels in distinct cellular compartments

• Ex vivo tissue culture:

  • Maintain tissue explants in culture medium with phosphatase inhibitors

  • Treat with estrogens or pathway modulators

  • Monitor dynamic changes in ESR2 phosphorylation

• Single-cell analysis:

  • Employ single-cell Western blotting or mass cytometry

  • Characterize cell-to-cell variability in phosphorylation status

  • Correlate with cell phenotype and functional state

These approaches enable detailed characterization of tissue-specific and cell-type-specific regulation of ESR2 phosphorylation that may be missed in whole-tissue analyses.

What are common technical challenges when detecting phospho-ESR2 and how can they be overcome?

Several technical challenges may arise when working with Phospho-ESR2 (S87) Antibody:

• Low signal intensity:

  • Increase antibody concentration (use 1:500 dilution instead of 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use signal enhancement systems (highly sensitive ECL substrates)

  • Immunoprecipitate ESR2 before Western blotting to concentrate the target

• High background:

  • Use 5% BSA instead of milk for blocking (milk contains phosphoproteins)

  • Include 0.1% Tween-20 in wash buffers

  • Increase number and duration of wash steps

  • Try alternative blocking agents (casein, commercial blockers)

• Multiple bands:

  • Validate specificity with blocking peptide competition

  • Use gradient gels for better resolution

  • Include positive control samples with known phospho-ESR2 expression

  • Consider alternative ESR2 isoforms that may be detected

• Inconsistent results:

  • Standardize lysate preparation protocol

  • Use fresh phosphatase inhibitors

  • Maintain consistent sample handling time

  • Prepare all samples simultaneously when possible

These optimizations can significantly improve detection reliability and signal specificity.

How can researchers differentiate between specific and non-specific signals when using Phospho-ESR2 (S87) Antibody?

Differentiating specific from non-specific signals requires systematic validation:

• Validation experiments:

  • Blocking peptide competition: Compare antibody alone vs. antibody pre-incubated with phosphopeptide

  • Dephosphorylation control: Treat duplicate samples with lambda phosphatase

  • Genetic validation: Use ESR2 knockout/knockdown samples as negative controls

• Signal characteristics to evaluate:

  • Molecular weight: True ESR2 signal should appear at 59-63 kDa

  • Stimulus responsiveness: Specific signal should increase with estrogen stimulation

  • Inhibitor sensitivity: Signal should decrease with appropriate kinase inhibitors

• Quantitative assessment:

  • Calculate signal-to-noise ratio for different experimental conditions

  • Compare staining patterns across multiple tissues with known ESR2 expression levels

  • Evaluate subcellular localization (phospho-ESR2 should show nuclear enrichment)

This multi-faceted approach establishes confidence in the specificity of detected signals.

What methodological approaches can address low abundance of phosphorylated ESR2 in experimental samples?

Low abundance of phosphorylated targets requires specialized enrichment strategies:

• Protein concentration techniques:

  • Immunoprecipitation of total ESR2 followed by phospho-detection

  • Phosphoprotein enrichment columns prior to Western blotting

  • TCA precipitation to concentrate proteins from dilute samples

• Signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry/immunofluorescence

  • Enhanced chemiluminescence systems for Western blotting

  • Proximity ligation assay for in situ detection with increased sensitivity

• Sample preparation optimization:

  • Use phosphatase inhibitor cocktails at higher concentrations

  • Reduce time between sample collection and processing

  • Optimize cell lysis conditions to maximize extraction efficiency

• Detection system selection:

  • Use highly sensitive digital imaging systems

  • Employ fluorescently-labeled secondary antibodies for multiplexing

  • Consider specialized high-sensitivity ELISA formats

These approaches can significantly improve detection of low-abundance phosphorylated ESR2 in various experimental systems.

How should researchers quantitatively analyze phospho-ESR2 levels across experimental conditions?

Robust quantitative analysis requires systematic approaches:

• Western blot densitometry:

  • Use digital imaging systems with wide dynamic range

  • Ensure exposure times avoid signal saturation

  • Perform background subtraction with local background sampling

  • Normalize phospho-ESR2 signal to total ESR2 and then to loading control

• Immunohistochemistry quantification:

  • Use digital pathology software for unbiased analysis

  • Quantify both staining intensity and percentage of positive cells

  • Develop scoring system (e.g., H-score = intensity × percentage)

  • Include reference standards in each batch for cross-comparison

• Statistical analysis:

  • Calculate fold changes relative to control conditions

  • Apply appropriate statistical tests based on data distribution

  • Consider non-parametric tests for ratio data (phospho/total ratios)

  • Use multiple comparison corrections for experiments with many conditions

• Data presentation:

  • Include representative images alongside quantitative data

  • Present both raw values and normalized ratios

  • Use box plots or violin plots to show data distribution

  • Indicate biological and technical replicates clearly

This systematic approach ensures reliable quantitative interpretation of phosphorylation changes across experimental conditions.

How can phospho-ESR2 data be integrated with other molecular data for comprehensive pathway analysis?

Integrative analysis enhances mechanistic insights:

• Multi-omics integration approaches:

  • Correlate phospho-ESR2 levels with gene expression profiles

  • Integrate with proteomics data on interacting partners

  • Map onto known signaling networks using pathway databases

• Computational tools:

  • Use Phosprof or similar phosphorylation databases for pathway mapping

  • Apply enrichment analysis to identify coordinated pathway activation

  • Employ network visualization tools to represent complex interactions

• Validation of predicted relationships:

  • Design targeted experiments to test computational predictions

  • Use pharmacological or genetic perturbations of key nodes

  • Monitor effects on phospho-ESR2 and downstream events

• Temporal dynamics integration:

  • Align time courses of phosphorylation with transcriptional changes

  • Establish cause-effect relationships through time-resolved data

  • Develop mathematical models of ESR2 signaling dynamics

This integrative approach places phospho-ESR2 data in broader biological context and enhances mechanistic understanding of its functional role.

How can contradictory findings regarding ESR2 phosphorylation be reconciled across different experimental systems?

Reconciling disparate findings requires systematic evaluation:

• Methodological comparison table:

  • Create a comprehensive comparison of experimental methods

  • Document antibodies, concentrations, detection systems used

  • Evaluate differences in sample preparation procedures

  • Assess timing of measurements relative to stimulation

• Biological variables assessment:

  • Compare cell types, tissues, and species used

  • Evaluate differences in ESR2 expression levels and isoforms

  • Consider cell culture conditions and microenvironment

  • Analyze potential differences in basal phosphorylation states

• Experimental validation:

  • Reproduce key experiments under standardized conditions

  • Use multiple antibodies and detection methods

  • Employ genetic tools (S87A mutants) to confirm specificity

  • Consider collaborations between labs with contradictory findings

• Context-dependent interpretation:

  • Develop models that incorporate context-specific regulation

  • Identify factors that may switch phosphorylation outcomes

  • Consider cell-type-specific or tissue-specific regulatory mechanisms

This systematic approach can transform seemingly contradictory findings into a more nuanced understanding of context-dependent regulation.

How might single-cell analysis technologies advance our understanding of ESR2 phosphorylation heterogeneity?

Single-cell technologies offer new research frontiers:

• Single-cell Western blotting:

  • Analyze phospho-ESR2 levels in individual cells

  • Correlate with cell cycle stage or differentiation status

  • Identify rare cell populations with unique phosphorylation profiles

• Mass cytometry (CyTOF):

  • Multiplex phospho-ESR2 with dozens of other cellular markers

  • Create high-dimensional maps of signaling states

  • Apply clustering algorithms to identify cell subpopulations

• Single-cell phosphoproteomics:

  • Profile the broader phosphoproteome in ESR2-expressing cells

  • Identify coordinated phosphorylation networks at single-cell resolution

  • Discover new signaling relationships invisible in bulk analysis

• Spatial transcriptomics integration:

  • Correlate phospho-ESR2 patterns with spatial gene expression

  • Map phosphorylation status to tissue architecture

  • Identify niche-dependent regulation of ESR2 phosphorylation

These approaches can reveal cell-to-cell variability in ESR2 phosphorylation that may have important functional consequences previously masked in population-averaged measurements.

What potential role might phospho-ESR2 (S87) play as a biomarker in hormone-responsive diseases?

Biomarker development requires systematic investigation:

• Clinical correlation studies:

  • Analyze phospho-ESR2 (S87) levels in patient samples

  • Correlate with clinical parameters and treatment outcomes

  • Evaluate prognostic and predictive value

• Technical requirements for clinical translation:

  • Develop standardized protocols for clinical sample processing

  • Validate antibody performance across multiple laboratories

  • Establish quantitative cutoff values for clinical decision-making

• Combination biomarker approaches:

  • Integrate phospho-ESR2 with other established biomarkers

  • Develop multiplexed assays for comprehensive profiling

  • Create predictive algorithms incorporating multiple parameters

• Functional validation:

  • Establish mechanistic links between phosphorylation status and disease progression

  • Determine whether phosphorylation status predicts treatment response

  • Investigate phospho-ESR2 as a potential therapeutic target

This research trajectory could establish phospho-ESR2 (S87) as a clinically relevant biomarker for stratification or treatment selection in hormone-responsive diseases.

How can new technologies in antibody engineering improve phospho-ESR2 detection specificity and sensitivity?

Emerging antibody technologies offer potential advancements:

• Recombinant antibody formats:

  • Single-chain variable fragments (scFvs) for improved tissue penetration

  • Bispecific antibodies recognizing both ESR2 and the phospho-epitope

  • Intrabodies for live-cell imaging of phosphorylation events

• Synthetic phospho-specific binders:

  • Phospho-binding domains fused to detection tags

  • Engineered protein scaffolds with phospho-specificity

  • Aptamer-based detection systems for phospho-ESR2

• Proximity-based detection systems:

  • Split-reporter systems that activate upon phosphorylation

  • FRET-based biosensors for real-time monitoring

  • Bioluminescence resonance energy transfer (BRET) systems

• Nanobody technology:

  • Develop phospho-specific nanobodies (VHH fragments)

  • Create intracellular nanobody sensors for live imaging

  • Design nanobody-based proximity ligation systems

These technological advances could overcome current limitations in phospho-ESR2 detection and enable new experimental approaches for studying dynamic phosphorylation events in living systems.