Phospho-ESR1 (Ser167) Antibody

What exactly does the Phospho-ESR1 (Ser167) Antibody detect?

The Phospho-ESR1 (Ser167) Antibody specifically detects endogenous levels of Estrogen Receptor alpha (ESR1) protein only when phosphorylated at the Serine 167 position . This specificity is crucial for studying the phosphorylation state of the receptor independent of total receptor expression levels. The antibody recognizes a specific epitope around the phosphorylated Ser167 residue, typically within the amino acid range 136-185 of the human ESR1 protein . This high specificity allows researchers to distinguish between phosphorylated and non-phosphorylated forms of the receptor, enabling detailed studies of ESR1 activation states in various biological contexts.

The antibody's specificity is typically assured through careful design of the immunogen, which is a synthesized peptide derived from the human Estrogen Receptor-alpha sequence surrounding the phosphorylation site of Ser167 . Most commercially available antibodies against this epitope are polyclonal antibodies raised in rabbits, purified through affinity chromatography using epitope-specific immunogens to ensure high specificity for the phosphorylated form of the receptor .

What is the biological significance of ESR1 phosphorylation at Serine 167?

Phosphorylation of ESR1 at Serine 167 represents a critical post-translational modification that regulates estrogen receptor function and signaling. This phosphorylation event is particularly significant as it probably enhances the transcriptional activity of the receptor . In the context of breast cancer, phosphorylation at Ser167 has emerged as a potential predictive biomarker for response to endocrine therapy, as indicated by clinical studies . This modification appears to be part of the complex regulatory network that modulates estrogen receptor activity in both normal physiology and pathological conditions.

The phosphorylation of Ser167 is mediated by various kinases including cyclin A/CDK2 and CK1, representing integration points between estrogen signaling and other cellular pathways . Additionally, this phosphorylation site can be affected by growth factor signaling networks, providing a mechanism for crosstalk between estrogen receptor function and other signaling cascades. The dynamic regulation of this modification contributes to the context-dependent activity of estrogen receptors in different tissues and under different physiological or pathological conditions.

How does ESR1 Ser167 phosphorylation relate to other post-translational modifications of the receptor?

ESR1 undergoes multiple post-translational modifications that collectively determine its activity, stability, and cellular localization. Phosphorylation at Ser167 is just one of several phosphorylation events that regulate receptor function. Other key phosphorylation sites include Ser118, which is also extensively studied in the context of receptor activation . These phosphorylation events may occur independently or in coordination, creating a complex code that fine-tunes receptor activity in response to various stimuli.

Beyond phosphorylation, ESR1 is subject to other modifications including glycosylation, ubiquitination, palmitoylation, and methylation . For instance, ESR1 can be ubiquitinated by STUB1/CHIP and deubiquitinated by OTUB1, processes that regulate receptor stability and turnover. The receptor can also be dimethylated by PRMT1 at Arg-260, which may influence its cellular localization . Palmitoylation by ZDHHC7 and ZDHHC21 facilitates plasma membrane targeting and is required for rapid intracellular signaling via ERK and AKT kinases . These diverse modifications form an intricate regulatory network that modulates ESR1 function in different cellular contexts and in response to various stimuli.

What are the optimal conditions for using Phospho-ESR1 (Ser167) Antibody in Western blotting?

When using Phospho-ESR1 (Ser167) Antibody for Western blotting, researchers should optimize several key parameters to ensure specific and sensitive detection. Based on manufacturer recommendations, appropriate dilution ranges typically fall between 1:500 and 1:2000 . The antibody performs optimally when detecting the 66 kDa ESR1 protein (calculated molecular weight) , although researchers should be aware that alternative splicing can generate multiple isoforms of ESR1 with different molecular weights .

Sample preparation is crucial for maintaining phosphorylation status. Tissues or cells should be lysed in buffers containing phosphatase inhibitors to prevent dephosphorylation during processing. Use of PVDF membranes is recommended, as demonstrated in published protocols . For enhanced sensitivity, researchers may employ enhanced chemiluminescence substrate mixtures such as SuperSignal WestPico Chemiluminescent Substrate . Proper blocking (typically with 5% BSA in TBST rather than milk, which contains phosphatases) and appropriate controls are essential for result validation. A control detecting total ESR1 should be run in parallel to normalize phosphorylation signals to total protein levels, enabling accurate quantification of the phosphorylation state.

What are the recommended protocols for immunohistochemical detection of phospho-ESR1 (Ser167)?

For immunohistochemical applications, the Phospho-ESR1 (Ser167) Antibody can be used to detect phosphorylated receptor in both paraffin-embedded (IHC-p) and frozen tissue sections (IHC-f) . Based on published research and manufacturer guidelines, dilution ratios typically range from 1:50 to 1:300 for IHC applications . The choice of dilution should be empirically determined for each experimental system and may vary depending on the specific antibody formulation, tissue type, and fixation method.

Antigen retrieval is a critical step for detecting phospho-epitopes in fixed tissues. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0) is commonly employed. Tissue sections should be deparaffinized, rehydrated, and subjected to antigen retrieval before blocking endogenous peroxidase activity and non-specific binding sites. For detection systems, researchers can use horseradish peroxidase-conjugated secondary antibodies followed by DAB chromogen for visualization . Appropriate positive and negative controls should always be included, such as tissues known to express phosphorylated ESR1 and sections where the primary antibody is omitted. For quantification, scoring systems based on staining intensity and percentage of positive cells can be employed, as is common in breast cancer research.

How can researchers validate the specificity of Phospho-ESR1 (Ser167) Antibody?

Validating antibody specificity is crucial for ensuring reliable research results, particularly with phospho-specific antibodies. Several complementary approaches can be employed to confirm the specificity of Phospho-ESR1 (Ser167) Antibody. First, researchers should conduct peptide competition assays, where the antibody is pre-incubated with the phosphorylated peptide used as the immunogen before application to the sample. This should abolish specific binding if the antibody is truly phospho-specific .

Another critical validation method involves using phosphatase treatment controls. Samples can be divided and one portion treated with lambda phosphatase to remove phosphorylation groups. A true phospho-specific antibody will show diminished or absent signal in the phosphatase-treated sample compared to the untreated control. Additionally, researchers can use cellular models where phosphorylation at Ser167 is either enhanced through treatment with estradiol or growth factors known to activate this site, or reduced through kinase inhibitors that block the relevant phosphorylation pathways.

For genetic validation, siRNA or CRISPR knockout of ESR1 should eliminate all signal when using the phospho-specific antibody in ESR1-expressing cells. Finally, comparing results across different detection methods (e.g., Western blot, IHC, IF) and different antibody clones targeting the same phospho-epitope can provide further confidence in antibody specificity and experimental results.

How does phosphorylation of ESR1 at Ser167 relate to breast cancer prognosis and treatment response?

Phosphorylation of ESR1 at Serine 167 has emerged as a significant biomarker in breast cancer research with important clinical implications. Research has demonstrated that this specific phosphorylation event is predictive of response to endocrine therapy in patients with hormone-receptor-positive breast cancer . The phosphorylation status at this site provides valuable information beyond mere expression levels of the estrogen receptor, potentially helping to stratify patients who are more likely to benefit from hormonal treatments.

Studies examining primary breast carcinomas from patients with metastatic disease have shown correlations between phospho-ESR1 (Ser167) status and clinical outcomes. Patients with tumors exhibiting higher levels of ESR1 phosphorylation at Ser167 may show different response patterns to various endocrine therapies, including selective estrogen receptor modulators (SERMs) and aromatase inhibitors . This phosphorylation event appears to be linked to the activation of specific downstream pathways that influence tumor behavior and therapeutic response. The mechanism likely involves altered transcriptional activity of the receptor when phosphorylated at this site, as phosphorylation is known to enhance transcriptional function .

What techniques can be combined with Phospho-ESR1 (Ser167) Antibody for comprehensive signaling pathway analysis?

To comprehensively analyze signaling pathways involving phosphorylated ESR1, researchers can employ multiple complementary techniques alongside Phospho-ESR1 (Ser167) Antibody detection. Multiplex immunofluorescence or immunohistochemistry allows simultaneous detection of phospho-ESR1 (Ser167) along with other phosphorylated signaling proteins, such as components of the PI3K/AKT or MAPK pathways, which may be involved in receptor phosphorylation. This approach can reveal spatial relationships between different activated pathways within tissue sections.

Chromatin immunoprecipitation (ChIP) assays using Phospho-ESR1 (Ser167) Antibody can identify genomic binding sites specifically occupied by the phosphorylated form of the receptor, potentially revealing distinct transcriptional programs regulated by this active form. Combining this with RNA-seq analysis provides comprehensive insights into the transcriptional consequences of ESR1 phosphorylation at Ser167. Additionally, proximity ligation assays (PLA) can detect interactions between phospho-ESR1 (Ser167) and specific coregulatory proteins, helping to elucidate how phosphorylation affects protein-protein interactions.

Phosphoproteomics approaches, including mass spectrometry-based techniques, can map broader phosphorylation networks that are active concurrently with ESR1 Ser167 phosphorylation. This systems-level analysis can reveal how ESR1 phosphorylation fits into larger signaling networks and identify potential therapeutic targets within these pathways. Reverse phase protein arrays (RPPA) offer a high-throughput method to analyze multiple phosphorylated proteins simultaneously across many samples, facilitating correlation analyses between phospho-ESR1 (Ser167) and other signaling nodes.

What factors might affect phosphorylation status of ESR1 during sample preparation?

Maintaining the phosphorylation status of ESR1 during sample preparation is critical for accurate analysis. Several factors can significantly impact phosphorylation detection. First and foremost, the time between tissue acquisition or cell harvesting and sample fixation or lysis is crucial. Phosphorylation states can change rapidly post-mortem or after cell harvesting due to ongoing phosphatase activity. Immediate snap-freezing of tissues or rapid lysis of cells is recommended to preserve phosphorylation status .

The composition of lysis or fixation buffers is equally important. Buffers must contain effective phosphatase inhibitor cocktails that target multiple classes of phosphatases (e.g., serine/threonine phosphatases, tyrosine phosphatases, and acid phosphatases). Commercial phosphatase inhibitor cocktails are available and should be freshly added to buffers before use. Additionally, sample handling temperature affects enzymatic activity; all processing steps should ideally be performed at 4°C to minimize phosphatase activity.

For tissue samples, the fixation method and duration significantly impact phospho-epitope preservation. Extended formalin fixation can mask phospho-epitopes, necessitating optimization of antigen retrieval protocols. For cells in culture, treatment conditions prior to harvesting, including serum starvation, growth factor stimulation, or drug treatments, can dramatically alter ESR1 phosphorylation states. Consistent and well-documented sample handling procedures are essential for reliable and reproducible phosphorylation analysis across experiments.

What are common causes of false positive or negative results when using Phospho-ESR1 (Ser167) Antibody?

Several technical and biological factors can lead to inaccurate results when working with Phospho-ESR1 (Ser167) Antibody. False positive results may arise from non-specific binding of the antibody to other phosphorylated proteins with similar epitopes. This can be particularly problematic with polyclonal antibodies, which contain multiple antibody clones with varying specificities . Cross-reactivity with other phosphorylated serine residues in similar sequence contexts should be carefully considered when interpreting results.

Insufficient blocking or inappropriate blocking agents can also contribute to background signal and false positives. When detecting phospho-epitopes, researchers should typically use BSA for blocking rather than milk, as milk contains phosphatases that might dephosphorylate the target protein during incubation steps. Additionally, endogenous peroxidase activity in tissues or cells can generate false positive signals in peroxidase-based detection systems if not properly quenched.

False negative results frequently stem from inadequate preservation of phosphorylation during sample preparation, as discussed previously. Additionally, epitope masking due to protein-protein interactions or conformational changes may prevent antibody binding. This can be particularly relevant for nuclear receptors like ESR1, which form complexes with numerous coregulatory proteins. Insufficient antigen retrieval in fixed tissues is another common cause of false negatives. The optimal antigen retrieval method may need to be empirically determined for each tissue type and fixation protocol.

How can the dilution ranges of Phospho-ESR1 (Ser167) Antibody be optimized for different applications?

The optimal antibody dilution varies significantly across different applications and must be empirically determined for each experimental system. According to manufacturer recommendations and published research, typical dilution ranges include: Western blotting (1:500-1:2000), immunohistochemistry (1:100-1:300), immunofluorescence (1:50-200), and ELISA (1:20000) . These ranges serve as starting points, and researchers should perform titration experiments to determine the optimal concentration for their specific samples and conditions.

For Western blotting, a dilution series should be tested while keeping all other parameters constant. The optimal dilution provides a strong specific signal for the target protein (66 kDa for ESR1) with minimal background. For IHC applications, optimization should evaluate different antibody concentrations alongside various antigen retrieval methods, as these factors interact to determine staining quality . A good IHC protocol produces distinct nuclear staining in ER-positive cells with minimal cytoplasmic or stromal background.

When optimizing immunofluorescence protocols, both antibody concentration and incubation conditions (time and temperature) should be systematically varied. Lower dilutions (higher antibody concentrations) may be required for detecting low-abundance phosphorylated proteins, but this increases the risk of non-specific binding. Including appropriate positive controls (samples known to contain phosphorylated ESR1 at Ser167) and negative controls (samples treated with phosphatases or from ESR1-negative tissues) is essential for accurate protocol optimization.

How can Phospho-ESR1 (Ser167) Antibody be used to study endocrine resistance mechanisms?

The Phospho-ESR1 (Ser167) Antibody serves as a powerful tool for investigating mechanisms of endocrine resistance in breast cancer, a significant clinical challenge affecting many patients with ER-positive disease. Research has demonstrated that phosphorylation of ESR1 at Ser167 is predictive of response to endocrine therapy, suggesting its involvement in treatment sensitivity pathways . By using this antibody to monitor phosphorylation levels in cell line models and patient samples before and after development of resistance, researchers can identify changes in ESR1 activation states associated with therapeutic failure.

The antibody can be employed in time-course experiments examining how ESR1 phosphorylation patterns change during the development of resistance to various endocrine therapies, including tamoxifen, fulvestrant, or aromatase inhibitors. Such studies may reveal whether altered phosphorylation is an early event in resistance development or a consequence of other molecular changes. Additionally, combining phospho-ESR1 (Ser167) detection with analysis of upstream kinases known to target this site can illuminate which signaling pathways become dysregulated during resistance development.

In patient-derived xenograft (PDX) models or organoid cultures from endocrine-resistant tumors, the antibody can help assess the efficacy of novel combination therapies targeting both ESR1 and the kinases responsible for its phosphorylation. Immunohistochemical analysis of serial biopsies from patients undergoing endocrine therapy, using optimized protocols with this antibody , may identify early phosphorylation changes predictive of impending resistance, potentially allowing for timely therapeutic intervention.

What insights can Phospho-ESR1 (Ser167) Antibody provide about ESR1 function in non-breast tissues?

While estrogen receptor research has predominantly focused on breast cancer, ESR1 plays important roles in multiple tissues throughout the body. The Phospho-ESR1 (Ser167) Antibody can help illuminate tissue-specific phosphorylation patterns and their functional consequences in diverse physiological and pathological contexts. In the reproductive system beyond the breast, including ovaries, uterus, and placenta, patterns of ESR1 phosphorylation may reveal tissue-specific regulatory mechanisms and how these relate to reproductive disorders.

In the cardiovascular system, estrogen signaling through ESR1 has protective effects on vascular endothelium and heart muscle. Using Phospho-ESR1 (Ser167) Antibody in vascular tissue samples can help elucidate how sex-specific cardioprotection operates at the molecular level and how it might be therapeutically enhanced. Similarly, in bone, where estrogen prevents osteoporosis, tissue-specific patterns of ESR1 phosphorylation may explain differential responses to estrogen in osteoblasts versus osteoclasts.

The central nervous system contains widespread ESR1 expression with important roles in neuroprotection, cognition, and neuroendocrine regulation. Applying the antibody in brain tissue sections can map region-specific phosphorylation patterns and potentially reveal how these correlate with neuronal function or pathology. In neurodegenerative conditions with known sex differences in prevalence or progression, such as Alzheimer's disease, ESR1 phosphorylation status might provide insights into disease mechanisms and potential therapeutic approaches.

How does phosphorylation at Ser167 affect ESR1 interaction with transcriptional coregulators?

Phosphorylation of ESR1 at Ser167 has significant implications for how the receptor interacts with its extensive network of coregulatory proteins. This post-translational modification is known to enhance the transcriptional activity of the receptor , likely by modulating its ability to recruit specific coactivators to target gene promoters. The location of Ser167 within the receptor's structure places it in a region that can influence both DNA binding and interaction with transcriptional machinery.

Research suggests that phosphorylation at this site may enhance ESR1 binding to certain coactivators containing LXXLL motifs, such as members of the p160 family (SRC-1, SRC-2, SRC-3). These interactions are crucial for assembling the multiprotein complexes required for efficient transcriptional activation. Additionally, phosphorylation may affect the receptor's interaction with other transcription factors such as AP-1/c-Jun, c-Fos, ATF-2, Sp1, and Sp3, potentially influencing ERE-independent signaling pathways .

The relationship between ESR1 phosphorylation and coregulator recruitment can be studied using techniques such as chromatin immunoprecipitation (ChIP) with the Phospho-ESR1 (Ser167) Antibody, followed by analysis of associated coregulators. Co-immunoprecipitation experiments comparing wild-type ESR1 with phosphomimetic (S167E) and phospho-dead (S167A) mutants can further elucidate how this modification affects protein-protein interactions. Understanding these molecular mechanisms has significant implications for developing novel therapeutic approaches targeting specific ESR1 functions in various diseases.