GPER1 Antibody, Biotin conjugated

What is GPER1 and why is it significant for research?

GPER1 (G Protein-Coupled Estrogen Receptor 1) is a membrane-bound estrogen receptor that mediates rapid, non-genomic responses to estrogen. Unlike classical estrogen receptors (ERα and ERβ), GPER1 functions as a G protein-coupled receptor. The significance of GPER1 lies in its involvement in numerous physiological processes including cellular proliferation, migration, and inflammation, as well as its potential role in cancer development, particularly breast cancer. The receptor has attracted considerable interest in research due to its distinct signaling mechanisms and tissue-specific functions. GPER1 is a 375 amino acid protein (42.2 kDa) with complex subcellular localization patterns in the nucleus, cytoplasm, mitochondria, endoplasmic reticulum, Golgi, and plasma membrane, making it a multifaceted target for investigation . Understanding GPER1 biology is crucial for developing new therapeutic approaches for hormone-responsive conditions and cancers.

What are the key characteristics of GPER1 antibody, biotin conjugated?

The biotin-conjugated GPER1 antibody is a polyclonal antibody developed in rabbits that specifically targets human GPER1. The antibody is generated using a recombinant human GPER1 immunogen comprising amino acids 1-62 of the protein sequence . It has an IgG isotype and demonstrates high purity (>95%) following Protein G purification. The antibody is provided in liquid form in a buffer containing 0.01M PBS at pH 7.4, with 0.03% Proclin-300 as a preservative and 50% glycerol for stability . The biotin conjugation enables versatile detection strategies, particularly useful in amplification systems where enhanced sensitivity is required. This antibody has been primarily tested and validated for ELISA applications, making it suitable for quantitative detection of GPER1 in research samples . The specificity for human GPER1 should be considered when designing cross-species experiments, as reactivity with GPER1 orthologs from other species may vary.

How should GPER1 antibody, biotin conjugated be stored and handled to maintain its effectiveness?

Proper storage and handling of the biotin-conjugated GPER1 antibody is essential for maintaining its effectiveness and extending its usable lifespan. The antibody should be stored in aliquots at -20°C to prevent degradation and minimize the need for repeated freeze-thaw cycles . Because the antibody is biotin-conjugated, it is particularly sensitive to light exposure, which can degrade the biotin moiety and reduce detection capability. Therefore, protection from light during storage and handling is crucial . When working with the antibody, allow it to equilibrate to room temperature slowly before opening the vial to prevent condensation, which can introduce microbial contamination and accelerate degradation. After use, return the antibody promptly to -20°C. Prepare working dilutions only when needed and avoid storing diluted antibody solutions for extended periods. If any cloudiness or precipitation appears in the solution, this may indicate denaturation of the antibody, and such solutions should be centrifuged before use to remove particulates that might interfere with binding specificity .

What is the optimal protocol for using GPER1 antibody, biotin conjugated in ELISA?

When conducting ELISA with biotin-conjugated GPER1 antibody, a carefully optimized protocol enhances specificity and sensitivity. Begin by coating microplate wells with target capture antibody (if using sandwich ELISA) or directly with antigen (if using direct ELISA) overnight at 4°C. After washing with PBS containing 0.05% Tween-20, block non-specific binding sites with 5% non-fat milk or BSA in PBS for 1-2 hours at room temperature. For sample preparation, ensure proper extraction of GPER1 from cellular lysates or tissue samples using compatible lysis buffers that preserve protein conformation while minimizing interference from other cellular components. Incubate samples in coated wells (2-4 hours at room temperature or overnight at 4°C), followed by thorough washing to remove unbound material .

Add the biotin-conjugated GPER1 antibody at an empirically determined optimal dilution (starting recommendation is 1:500-1:2000, though optimal concentrations should be determined experimentally for each lot) . Incubate for 1-2 hours at room temperature with gentle agitation. After washing, add streptavidin-HRP conjugate and incubate for 30-60 minutes. Following a final wash step, develop with appropriate substrate (TMB for colorimetric detection) and stop the reaction with 2N H₂SO₄ before reading absorbance at 450nm. Include proper controls: positive control with known GPER1 protein, negative control without primary antibody, and background control without antigen. Standard curves using recombinant GPER1 protein help quantify your results accurately .

Can GPER1 antibody, biotin conjugated be used for applications beyond ELISA?

While the biotin-conjugated GPER1 antibody is primarily validated for ELISA applications, the biotin label makes it potentially adaptable for other techniques requiring signal amplification or alternative detection methods. Immunohistochemistry (IHC) could utilize this antibody with streptavidin-conjugated reporter systems, though antigen retrieval methods would need optimization due to GPER1's membrane localization and complex post-translational modifications including glycosylation . For immunofluorescence, the antibody could be detected using fluorophore-labeled streptavidin, providing visualization of GPER1's subcellular distribution across nuclear, cytoplasmic, mitochondrial, ER, Golgi, and membrane compartments .

What considerations are important when designing experiments to study GPER1-mediated signaling using this antibody?

When designing experiments to study GPER1-mediated signaling using biotin-conjugated GPER1 antibody, several critical factors must be considered. First, understand that GPER1 signaling is complex, potentially involving both ligand-dependent and constitutive activity pathways . Recent research highlights controversy regarding whether estrogen directly activates GPER1, with some studies suggesting that GPER1's interactome is not significantly affected by estrogen treatment, mirroring constitutive activity in functional assays . This necessitates careful experimental design with appropriate positive and negative controls, including selective agonists (G-1) and antagonists (G-15, G-36) to distinguish GPER1-specific signaling from effects mediated by classical estrogen receptors .

Consider GPER1's dynamic subcellular localization across nuclear, cytoplasmic, mitochondrial, ER, Golgi and membrane compartments when interpreting antibody-based detection results . The receptor undergoes internalization through clathrin-coated pits in a β-arrestin-independent manner, which may affect its availability for antibody binding in fixed versus live cell experiments . GPER1's post-translational modifications, particularly N-glycosylation in the extracellular N-terminal domain, can influence antibody binding efficiency and may vary across cell types . When studying GPER1 protein interactions, consider its PDZ domain interactions with proteins such as SAP97, AKAP5, PSD-95, PMCA4b, NHERF1, and RAMP3, which may form complexes that exhibit constitutive activity . Timing is also critical—GPER1 mediates rapid non-genomic responses, so kinetic experiments with appropriate time points are essential for capturing transient signaling events .

How can researchers overcome challenges in detecting GPER1 due to its post-translational modifications?

GPER1 undergoes significant post-translational modifications, particularly N-glycosylation in its extracellular N-terminal domain, which can mask epitopes and complicate antibody-based detection . To overcome these challenges, researchers should implement a multi-faceted approach. First, consider enzymatic deglycosylation treatments (PNGase F, Endo H) before antibody application to expose masked epitopes, though care must be taken to preserve protein conformation. Compare detection using antibodies targeting different epitopes—the biotin-conjugated antibody recognizes amino acids 1-62, which includes potential glycosylation sites , while antibodies targeting the C-terminal region may provide complementary information less affected by N-terminal modifications.

Validation through orthogonal methods is crucial: combine antibody-based detection with mass spectrometry to confirm protein identity and modification sites. Recombinant expression systems with controlled glycosylation patterns can serve as valuable controls. When studying GPER1 in different cell types or tissues, account for cell-specific glycosylation patterns that may alter antibody recognition efficiency. For western blotting applications, carefully analyze migration patterns—glycosylated GPER1 may appear at higher molecular weights than the predicted 42.2 kDa . Consider native gel conditions to preserve protein-modification integrity when possible. Finally, knockdown/knockout validation experiments provide definitive confirmation of antibody specificity regardless of post-translational modifications. This comprehensive approach ensures reliable detection and characterization of GPER1 despite its complex modification landscape .

What is the current understanding of GPER1's interactome and how can this antibody contribute to further research?

Recent proteomic analyses have significantly expanded our understanding of GPER1's interactome, with research combining APEX2-mediated proximity labeling and immunoprecipitation followed by mass spectrometry identifying 73 novel potential GPER1 interactors . Intriguingly, this interactome appears unaffected by estrogen treatment, consistent with observations of GPER1's constitutive activity in functional assays . Key interaction partners include proteins containing PDZ domains such as SAP97, AKAP5, PSD-95, PMCA4b, NHERF1, and RAMP3, which bind to GPER1's C-terminal PDZ motif . These interactions form complexes that may exhibit constitutive activity, regulating cAMP synthesis and maintaining GPER1 at the plasma membrane.

The biotin-conjugated GPER1 antibody can contribute to interactome research through several approaches. Its biotin tag enables streptavidin-based pulldown of GPER1 complexes for co-immunoprecipitation studies, potentially allowing identification of additional interaction partners. Proximity ligation assays (PLAs) can visualize and quantify specific protein-protein interactions in situ by combining this antibody with antibodies against suspected interaction partners. The antibody could also be used in protein array screening to identify novel binding partners from cell or tissue lysates. For validation of interactions, FRET or BRET analyses using the biotin-conjugated antibody with appropriate fluorescent streptavidin conjugates could assess direct protein-protein interactions in live cells. Additionally, this antibody may help clarify whether GPER1 interactions are constitutive or ligand-dependent, addressing the ongoing controversy regarding estrogen's direct role in GPER1 activation . These approaches could ultimately advance understanding of GPER1's signal transduction mechanisms and potentially assist in deorphanizing or redeorphanizing this receptor .

How does GPER1 signaling differ from classical estrogen receptor pathways, and what techniques using this antibody can differentiate them?

GPER1 signaling fundamentally differs from classical estrogen receptor (ER) pathways in several key aspects. While ERα and ERβ primarily function as nuclear receptors mediating genomic responses through direct DNA binding, GPER1 mediates rapid non-genomic responses through G-protein-coupled mechanisms . GPER1 signaling involves activation of adenylyl cyclase through heterotrimeric G proteins, intracellular calcium mobilization, MAPK (ERK1/2) activation, and crosstalk with epidermal growth factor receptor signaling . Additionally, GPER1 demonstrates constitutive activity that may be independent of estrogen binding, whereas classical ERs are primarily ligand-dependent transcription factors .

The biotin-conjugated GPER1 antibody can help differentiate these pathways through several strategic approaches. Researchers can employ immunocytochemistry with this antibody to visualize GPER1's distinct subcellular localization patterns across nuclear, cytoplasmic, mitochondrial, ER, Golgi, and membrane compartments, contrasting with the predominantly nuclear localization of activated classical ERs . Time-course experiments measuring rapid signaling events (seconds to minutes) characteristic of GPER1 versus slower genomic responses (hours) of classical ERs can be analyzed using phospho-specific antibodies against downstream targets like ERK1/2, with the biotin-conjugated GPER1 antibody confirming receptor expression and localization .

Selective pharmacological tools can be combined with antibody detection: G-1 (GPER1-selective agonist) versus ER-selective agonists (PPT for ERα, DPN for ERβ) can help distinguish pathway-specific responses . Co-immunoprecipitation studies using the biotin-conjugated antibody can identify GPER1-specific protein complexes that differ from classical ER interaction networks. RNA interference or CRISPR-based approaches targeting GPER1 versus classical ERs, followed by antibody-based verification of knockdown efficiency, can further separate the distinct contributions of each pathway. Together, these approaches leverage the biotin-conjugated GPER1 antibody to disambiguate the complex and overlapping estrogen signaling networks in experimental systems .

What are common technical challenges when working with GPER1 antibody, biotin conjugated, and how can they be resolved?

Researchers working with biotin-conjugated GPER1 antibody may encounter several technical challenges that require systematic troubleshooting. High background signal is a common issue, potentially arising from excessive antibody concentration, insufficient blocking, or endogenous biotin in samples. To resolve this, optimize antibody dilution (starting with manufacturer recommendations and titrating as needed), extend blocking time with biotin-free blocking reagents, and consider using avidin/biotin blocking kits to mask endogenous biotin . Weak or absent signal may result from insufficient antigen retrieval, epitope masking due to GPER1's post-translational modifications (particularly glycosylation), or antibody degradation. Address this by optimizing antigen retrieval protocols, considering enzymatic deglycosylation treatments where appropriate, and ensuring proper antibody storage conditions (aliquoted at -20°C, protected from light, and minimizing freeze-thaw cycles) .

Non-specific binding can occur due to GPER1's structural similarity to other G-protein coupled receptors. Validate specificity using positive and negative controls, including GPER1 knockdown/knockout samples and recombinant GPER1 protein. Signal variability between experiments may stem from inconsistent handling of the biotin-conjugated antibody. Standardize experimental protocols, prepare fresh working dilutions for each experiment, and maintain consistent incubation times and temperatures . If detection problems persist, consider the possibility of sample-specific issues such as protein degradation or epitope masking. Fresh sample preparation, alternative lysis buffers, and comparison with different GPER1 antibodies targeting distinct epitopes can help troubleshoot these more complex issues. Each of these approaches should be systematically evaluated and documented to develop optimal protocols for specific experimental contexts .

How can researchers validate the specificity of their results when using GPER1 antibody, biotin conjugated?

Validating the specificity of results obtained with biotin-conjugated GPER1 antibody requires a comprehensive multi-method approach. Begin with positive and negative control samples: recombinant human GPER1 protein serves as a positive control, while cell lines with confirmed GPER1 knockdown/knockout provide essential negative controls . Peptide competition assays, where the antibody is pre-incubated with excess immunizing peptide (amino acids 1-62 of human GPER1) before application to samples, can confirm binding specificity—signals that disappear after peptide competition represent specific recognition .

Cross-validation using multiple antibodies targeting different GPER1 epitopes provides critical confirmation of results. If the biotin-conjugated antibody (targeting amino acids 1-62) produces patterns similar to antibodies targeting other regions, specificity is supported . Orthogonal detection methods strengthen validation: combine antibody-based detection with mRNA expression analysis (qPCR, RNA-seq) to confirm correlation between protein and transcript levels. For functional validation, pair antibody detection with GPER1-selective pharmacological tools (agonist G-1, antagonists G-15/G-36) to confirm that detected protein responds appropriately to these compounds .

Species specificity must be considered since this antibody is validated for human GPER1 . When working with non-human samples, sequence alignment analysis of the target epitope region (amino acids 1-62) can predict cross-reactivity. Size validation is also important—GPER1 has a predicted molecular weight of 42.2 kDa, but glycosylation and other post-translational modifications may alter migration patterns in gel-based applications . Finally, comprehensive reporting of validation steps in publications enhances reproducibility and confidence in results. This methodical validation process ensures that findings genuinely reflect GPER1 biology rather than technical artifacts .

What considerations are important when comparing results between different GPER1 antibodies in multi-antibody approaches?

When implementing multi-antibody approaches to study GPER1, researchers must carefully consider several factors that influence result interpretation. Epitope differences are primary considerations—the biotin-conjugated antibody targets amino acids 1-62 of human GPER1 , while other commercially available antibodies may target C-terminal, intracellular loops, or other N-terminal regions. These different epitopes experience varying accessibility depending on GPER1's conformational state, membrane integration, and protein-protein interactions . Post-translational modifications significantly impact detection—N-glycosylation in the N-terminal domain targeted by this biotin-conjugated antibody may mask epitopes, resulting in differential detection efficiency compared to antibodies targeting unmodified regions .

Antibody format comparisons require careful standardization—biotin conjugation enables signal amplification through streptavidin systems, potentially yielding higher sensitivity than directly conjugated antibodies but potentially different background characteristics . When comparing monoclonal versus polyclonal antibodies (the biotin-conjugated antibody being polyclonal), recognize that polyclonals typically provide broader epitope recognition but potentially increased cross-reactivity compared to the single-epitope specificity of monoclonals .

Standardized protocols are essential when comparing different antibodies—maintain identical sample preparation, blocking conditions, incubation times/temperatures, and detection systems whenever possible. When this isn't feasible, perform parallel optimization for each antibody. Quantification methods must account for different detection efficiencies—absolute signal intensity cannot be directly compared between different antibodies without appropriate calibration. Instead, focus on relative changes under experimental conditions for each antibody separately. Document all differences in methodology and antibody characteristics when publishing results to ensure proper interpretation by the scientific community. These considerations enable researchers to leverage complementary information from multiple antibodies while avoiding misinterpretation of technical differences as biological phenomena .

How is GPER1 research contributing to our understanding of estrogen-related cancers?

GPER1 research has significantly expanded our understanding of estrogen-related cancers, particularly breast cancer, by revealing non-classical estrogen signaling mechanisms that may influence cancer development and progression. Studies have established associations between GPER1 expression and various cancer types, with particular attention to breast cancer where its expression potentially serves as a prognostic marker . The receptor's complex signaling mechanisms—involving rapid activation of MAPK pathways, calcium mobilization, and crosstalk with epidermal growth factor receptor—provide additional layers to our understanding of how estrogen influences cancer cell proliferation, migration, and invasion beyond classical genomic pathways .

Intriguingly, GPER1 can mediate responses to selective estrogen receptor modulators (SERMs) like tamoxifen, potentially explaining some therapeutic resistance mechanisms in breast cancer treatment. Recent proteomic analyses have identified novel GPER1 interactors that may represent previously unrecognized signaling nodes in cancer biology . The controversial question of whether estrogen directly activates GPER1 has significant implications for cancer research—recent findings suggesting GPER1's constitutive activity and estrogen-independent interactome challenge the traditional view of estrogen-dependent signaling in cancer .

The biotin-conjugated GPER1 antibody contributes to cancer research by enabling detection of GPER1 expression in patient samples and experimental models, facilitating studies on receptor localization and expression patterns across cancer stages. Future directions include exploring GPER1-selective compounds as potential therapeutic agents, investigating GPER1's role in cancer stem cell biology, and clarifying how GPER1 signaling networks interact with tumor microenvironments. As our understanding evolves, GPER1-targeted approaches may offer novel therapeutic strategies for patients with estrogen-related cancers, particularly those resistant to conventional endocrine therapies .

What is the current controversy surrounding GPER1 activation by estrogen, and how can researchers address this question?

A significant controversy in GPER1 research centers on whether estrogen (17β-estradiol, E2) directly binds to and activates GPER1. Despite early characterization of GPER1 as a membrane estrogen receptor, recent investigations have raised substantial doubts . Several key points define this controversy: studies using the PathHunter β-arrestin recruitment technology failed to demonstrate β-arrestin recruitment upon E2 stimulation in GPER1-expressing HEK293T cells, challenging the classical GPCR activation model . Additionally, the ERα isoform ER-α36 was shown to mediate non-genomic responses through high-affinity binding to both E2 and the GPER1-specific agonist G-1 in certain cell lines, suggesting potential misattribution of signaling effects . Furthermore, proteomic analyses revealed that GPER1's interactome remains unaffected by estrogen treatment, mirroring the receptor's constitutive activity in functional assays with a Rac1 sensor .

Researchers can address this controversy through several methodological approaches. Direct binding studies using purified GPER1 protein and radiolabeled estrogen with appropriate controls can assess physical interaction. Comparing signaling responses in cell models with selective knockdown/knockout of GPER1 versus classical ERs can help delineate pathway-specific effects. CRISPR-engineered point mutations in potential ligand-binding domains of GPER1 may identify critical residues for estrogen responsiveness. Single-cell analysis techniques can address the heterogeneity in GPER1 responses observed across different cell types and contexts .

The biotin-conjugated GPER1 antibody could contribute to these investigations by facilitating immunoprecipitation of GPER1 complexes for binding studies or pull-down experiments to examine ligand-dependent versus constitutive interactions. As emphasized in recent literature, novel tools and approaches are needed to clarify whether GPER1 directly binds estrogen or other ligands and how signal transduction occurs—this represents a critical knowledge gap that must be addressed to advance understanding of estrogen signaling mechanisms .

What are promising future research directions for GPER1, and how might this antibody facilitate those studies?

Several promising future research directions are emerging in the GPER1 field that may be facilitated by the biotin-conjugated GPER1 antibody. The comprehensive characterization of GPER1's interactome has opened new avenues for understanding its signaling mechanisms. Recent proteomic analyses have identified 73 novel potential GPER1 interactors, including proteins like PRKCSH, CLPTM1, GANAB, and STIM1 . These interactions warrant detailed investigation to elucidate their functional significance in different cellular contexts. The biotin-conjugated antibody could facilitate co-immunoprecipitation experiments to confirm these interactions and examine how they change under various physiological and pathological conditions.

The question of GPER1's ligand specificity and activation mechanisms remains controversial. Innovative approaches combining structural biology, computational modeling, and functional genomics are needed to resolve whether GPER1 directly binds estrogen or requires other molecular partners for activation . The biotin-conjugated antibody could support these efforts through affinity purification techniques to isolate GPER1 complexes for structural studies or binding assays. The emerging concept of GPER1's constitutive activity and its regulation through protein-protein interactions rather than classical ligand binding represents a paradigm shift warranting further investigation . This antibody could help identify regulatory factors that modulate this constitutive activity in different cellular compartments.

The role of GPER1 in physiological and pathological processes beyond cancer, including cardiovascular function, metabolism, and neurological processes, represents an expanding research frontier. Tissue-specific expression patterns of GPER1 in brain, placenta, and prostate suggest diverse functions that remain incompletely characterized . The biotin-conjugated antibody could support immunohistochemical studies across various tissues to map expression patterns and correlate with physiological functions. Ultimately, as suggested in recent research, efforts to "deorphanize or redeorphanize" GPER1 may fundamentally reshape our understanding of estrogen signaling pathways . This antibody provides an important tool in this ongoing scientific journey to clarify GPER1's true biological identity and function.

What are the key considerations for researchers beginning work with GPER1 antibody, biotin conjugated?

Researchers beginning work with biotin-conjugated GPER1 antibody should consider several key factors to ensure experimental success. First, recognize the antibody's specific characteristics: it is a rabbit polyclonal targeting amino acids 1-62 of human GPER1, with primary validation for ELISA applications . This specificity for human GPER1 necessitates careful consideration when designing cross-species experiments. Implement proper storage and handling procedures—aliquot upon receipt to minimize freeze-thaw cycles, store at -20°C, and protect from light exposure due to the biotin conjugation . When beginning experiments, optimize antibody concentration through careful titration rather than relying solely on recommended dilutions, as optimal concentrations may vary by application and sample type .

Validation is critical—include appropriate positive controls (recombinant human GPER1 protein) and negative controls (samples lacking GPER1 expression) in initial experiments to confirm specificity . Consider the complex post-translational modifications of GPER1, particularly N-glycosylation, which may affect epitope accessibility and detection efficiency . Familiarize yourself with the current controversies surrounding GPER1 biology, particularly regarding whether estrogen directly activates the receptor and its potential constitutive activity, as these will influence experimental design and interpretation .