Phospho-EGFR (S1070) Antibody

What is Phospho-EGFR (S1070) Antibody and what specific epitope does it recognize?

Phospho-EGFR (S1070) Antibody is a specialized research reagent designed to recognize and bind specifically to the phosphorylated form of Epidermal Growth Factor Receptor at the serine 1070 residue. These antibodies are typically generated in rabbits immunized with KLH conjugated synthetic phosphopeptides corresponding to amino acid residues surrounding S1070 of human EGFR . The specificity for this particular phosphorylation site enables researchers to distinguish between phosphorylated and non-phosphorylated forms of the receptor, providing insights into EGFR activation states. The antibody's ability to selectively recognize this specific post-translational modification is crucial for studying site-specific phosphorylation events that might regulate distinct aspects of EGFR biology. This selective recognition capability makes Phospho-EGFR (S1070) antibodies valuable tools in dissecting the complex regulation of EGFR signaling in normal and pathological contexts .

What are the technical specifications of commercially available Phospho-EGFR (S1070) antibodies?

Commercial Phospho-EGFR (S1070) antibodies are predominantly available as affinity-purified rabbit polyclonal antibodies with IgG isotype classification . These antibodies typically undergo a multi-step purification process, including protein A column purification followed by peptide affinity purification, to enhance their specificity for the phosphorylated target . The antibodies are generally supplied in PBS buffer containing 0.09% (W/V) sodium azide as a preservative . They are validated for Western blot applications with recommended dilution ranges varying between 1:100-1:1000, depending on the manufacturer and specific product . Some antibodies are additionally validated for ELISA and dot blot (DB) applications . The reactivity of these antibodies is primarily limited to human EGFR, with limited cross-reactivity testing for other species . The primary accession number commonly associated with the target protein is P00533, corresponding to human EGFR . Storage recommendations typically include maintaining the antibody at 2-8°C for short-term storage (up to 2 weeks) and at -20°C in small aliquots for long-term storage to prevent freeze-thaw cycles .

How does phosphorylation at S1070 relate to EGFR structure and function?

Phosphorylation of EGFR at S1070 represents one of multiple post-translational modifications that collectively regulate this receptor's activity and downstream signaling capabilities. EGFR functions as a receptor tyrosine kinase that binds ligands of the EGF family and activates several signaling cascades to convert extracellular stimuli into appropriate cellular responses . The S1070 residue is located in the cytoplasmic domain of EGFR, where phosphorylation events can influence receptor conformation, protein-protein interactions, and signaling output. While tyrosine phosphorylation sites like Y1068, Y1086, and Y1173 create well-characterized docking sites for adaptor proteins containing SH2 and PTB domains, serine phosphorylation at S1070 likely serves a regulatory function that modulates receptor activity through different mechanisms . Research using monoclonal antibodies against ADAM17 has demonstrated that inhibition of this protease reduces EGFR phosphorylation at multiple sites including S1070, suggesting this phosphorylation event contributes to EGFR-dependent tumor growth . The precise structural consequences of S1070 phosphorylation remain to be fully elucidated, but likely involve conformational changes that affect receptor dimerization, internalization, or interactions with downstream signaling components.

What are the optimal conditions for using Phospho-EGFR (S1070) Antibody in Western blotting?

For optimal Western blot results with Phospho-EGFR (S1070) Antibody, sample preparation is crucial and should begin with cell lysis in a buffer containing both protease and phosphatase inhibitors to preserve the phosphorylation status of EGFR. Proteins should be separated on 6-8% SDS-PAGE gels to achieve good resolution of EGFR, which has a molecular weight of approximately 170 kDa . Following transfer to a PVDF or nitrocellulose membrane, blocking should be performed with 5% BSA in TBST rather than milk (which contains phosphatases that could dephosphorylate the epitope). The primary antibody should be applied at the manufacturer-recommended dilution, typically 1:1000 for Western blot applications, and incubated overnight at 4°C to maximize specific binding . Thorough washing with TBST (at least 3-5 washes of 5-10 minutes each) is essential to reduce background. An HRP-conjugated anti-rabbit secondary antibody should be used at a dilution of approximately 1:5000-1:10000, followed by detection with enhanced chemiluminescence reagents. For phospho-specific antibodies like Phospho-EGFR (S1070), inclusion of appropriate controls is critical, including positive controls (EGF-stimulated cells), negative controls (untreated cells or EGFR inhibitor-treated cells), and phosphatase-treated samples to confirm phospho-specificity .

How should researchers design experiments to study the regulation of EGFR S1070 phosphorylation?

Designing robust experiments to study EGFR S1070 phosphorylation regulation requires a systematic approach addressing multiple aspects of this post-translational modification. Researchers should begin by selecting appropriate cellular models with detectable EGFR expression, such as A431 cells (which overexpress EGFR) or cancer cell lines known to depend on EGFR signaling . Time-course experiments following stimulation with EGFR ligands (EGF, TGFα, etc.) at physiologically relevant concentrations are essential to understand the kinetics of S1070 phosphorylation . To identify the kinases responsible for S1070 phosphorylation, researchers should employ both pharmacological inhibitors targeting candidate kinases and genetic approaches such as siRNA knockdowns or CRISPR knockout screens. Phosphatase involvement can be studied using phosphatase inhibitors of varying specificity, combined with phosphatase overexpression or knockdown experiments. To understand how S1070 phosphorylation coordinates with other post-translational modifications, multiplex assays simultaneously detecting multiple phosphorylation sites (e.g., Y1086, Y845, S1070, Y1173) should be implemented . Environmental factors potentially influencing S1070 phosphorylation, such as cell density, hypoxia, or nutrient availability, should be systematically varied while monitoring phosphorylation status using Western blotting with Phospho-EGFR (S1070) Antibody .

How can researchers effectively troubleshoot common issues when working with Phospho-EGFR (S1070) Antibody?

Effectively troubleshooting problems with Phospho-EGFR (S1070) Antibody requires systematic analysis of each experimental component. When facing weak or absent signals, researchers should first verify EGFR phosphorylation status by stimulating cells with fresh EGF and including positive control lysates from cells known to express phosphorylated EGFR . If phosphorylation is confirmed but detection remains poor, optimizing antibody concentration (testing a range around the recommended dilution), increasing protein loading, enhancing sensitivity with alternative detection systems, or trying a fresh antibody aliquot may resolve the issue . High background problems typically stem from insufficient blocking or washing; increasing BSA concentration in blocking buffer to 5%, extending blocking time to 2 hours, adding 0.1-0.3% Tween-20 to wash buffers, and performing additional washing steps can significantly improve signal-to-noise ratio. Multiple or unexpected bands might indicate degraded samples or cross-reactivity; always use fresh lysates with protease inhibitors, optimize gel percentage for better resolution, and compare patterns with total EGFR antibody results . For inconsistent results between experiments, standardize all aspects of the protocol including cell culture conditions, stimulation parameters, lysis methods, and Western blot procedures. If problems persist despite these measures, performing peptide competition assays can help determine whether the observed signal is specific to phosphorylated EGFR at S1070 or represents non-specific binding .

How should researchers quantify and normalize phospho-EGFR (S1070) signals in Western blots?

Proper quantification and normalization of phospho-EGFR (S1070) signals require rigorous methodology to ensure accurate interpretation of experimental results. Researchers should begin by capturing Western blot images using a digital imaging system with a linear detection range (e.g., CCD camera-based systems) rather than film, which has limited dynamic range . Densitometric analysis should be performed using specialized software (ImageJ, Image Lab, etc.) with consistent background subtraction methods applied across all samples. A multi-level normalization approach is recommended: first, normalize phospho-EGFR (S1070) signal to a loading control such as β-actin or GAPDH to account for variations in total protein loaded; second, normalize to total EGFR levels (from parallel blots or stripped and reprobed membranes) to distinguish changes in phosphorylation status from changes in receptor expression . When comparing phosphorylation changes across treatments or time points, results should be expressed as fold-change relative to appropriate control conditions. Technical replicates (multiple lanes of the same sample) and biological replicates (independent experimental repeats) are essential for statistical analysis, with a minimum of three biological replicates recommended for statistical validity. For more advanced analyses, researchers can employ multiplex detection systems that simultaneously measure multiple phosphorylation sites, allowing for normalization across sites and more comprehensive pathway analysis . When reporting results, both representative Western blot images and quantified, normalized data from multiple independent experiments should be presented, along with appropriate statistical analysis.

What insights can be gained by comparing phosphorylation at S1070 with other EGFR phosphorylation sites?

Comparative analysis of S1070 phosphorylation alongside other EGFR phosphorylation sites provides valuable insights into receptor regulation and signaling dynamics that cannot be obtained by studying any single site in isolation. Research has demonstrated that EGFR undergoes coordinated phosphorylation at multiple sites, including tyrosine residues (Y1086, Y845, Y1173) and serine/threonine residues like S1070 . Studies using monoclonal antibodies targeting ADAM17 have shown that inhibition of this protease simultaneously reduces EGFR phosphorylation at Y1086, Y845, S1070, and Y1173, suggesting these sites may be co-regulated in certain contexts . By monitoring phosphorylation patterns across multiple sites following various stimuli (different ligands, concentrations, or exposure times), researchers can construct temporal phosphorylation profiles that reveal the sequence of modification events. This approach can identify potential hierarchical relationships, where phosphorylation at one site enables or inhibits modifications at others. Differential susceptibility of phosphorylation sites to specific kinase or phosphatase inhibitors can reveal the enzymes responsible for each modification. Correlation analysis between site-specific phosphorylation levels and downstream pathway activation (MAPK, PI3K, STAT) can link particular phosphorylation events to specific signaling outcomes . Integrating these multi-site phosphorylation data with protein-protein interaction studies can further elucidate how different phosphorylation patterns create distinct "molecular barcodes" that recruit different sets of signaling proteins to activated EGFR.

How does S1070 phosphorylation correlate with EGFR-dependent biological outcomes?

Understanding the relationship between S1070 phosphorylation and EGFR-dependent biological outcomes requires integrating molecular data with functional assays across various experimental systems. Studies examining the effects of ADAM17 inhibition have demonstrated that treatments reducing EGFR phosphorylation at multiple sites, including S1070, correlate with tumor regression or stasis in EGFR-dependent tumor models, suggesting this phosphorylation event contributes to oncogenic signaling . The inhibitory activity of ADAM17-targeting antibodies like MEDI3622 correlates with EGFR activity across various tumor models and patient-derived xenografts, providing evidence that phosphorylation events including S1070 modification are functionally relevant in cancer contexts . While direct causative relationships between S1070 phosphorylation specifically and cellular outcomes remain to be fully established, correlation analyses between phosphorylation levels and phenotypic readouts (proliferation, migration, survival, differentiation) can begin to assign functional significance to this modification. Manipulation of S1070 phosphorylation status through site-directed mutagenesis (S1070A to prevent phosphorylation or S1070D/E to mimic constitutive phosphorylation) provides more direct evidence of its functional importance. Monitoring S1070 phosphorylation dynamics during processes such as receptor internalization, degradation, or recycling can reveal roles in trafficking regulation. In therapeutic contexts, tracking S1070 phosphorylation before and after treatment with EGFR-targeted drugs can identify potential biomarkers of response or resistance mechanisms .

How can Phospho-EGFR (S1070) Antibody be integrated into comprehensive signaling pathway analyses?

Integration of Phospho-EGFR (S1070) Antibody into comprehensive signaling pathway analyses requires sophisticated experimental designs that capture network-level properties of EGFR signaling. Multiplex Western blotting or Luminex-based assays incorporating Phospho-EGFR (S1070) Antibody alongside antibodies targeting other phosphorylated EGFR residues and downstream effectors (MAPK, PI3K, STAT) enables simultaneous monitoring of multiple signaling nodes under various conditions . This approach reveals how S1070 phosphorylation correlates with activation of specific downstream pathways. For broader analyses, researchers can employ proteomics approaches like iTRAQ-based mass spectrometry in conjunction with Phospho-EGFR (S1070) immunoprecipitation to obtain comprehensive phosphorylation profiles and identify proteins that specifically interact with EGFR when S1070 is phosphorylated . Dynamic analysis of signaling networks can be achieved through time-course experiments following various stimuli, with mathematical modeling of the resulting data to infer causal relationships and feedback mechanisms. Spatial information about signaling events can be captured through multiplexed immunofluorescence combining Phospho-EGFR (S1070) Antibody with other signaling component antibodies, revealing compartmentalization of signaling activities. Integration of these data with transcriptomic or metabolomic profiles creates multi-omic datasets that provide holistic views of cellular responses to EGFR activation. Network analysis and visualization tools can then be applied to these integrated datasets to identify signaling hubs, feedback loops, and potential vulnerabilities that might be targeted therapeutically in EGFR-dependent diseases .

What role does EGFR S1070 phosphorylation play in cancer pathogenesis and therapeutic resistance?

EGFR S1070 phosphorylation appears to be implicated in cancer pathogenesis and therapeutic resistance mechanisms, though its specific contributions continue to be elucidated through ongoing research. EGFR overexpression and hyperactivation are hallmarks of various cancers, with aberrant phosphorylation patterns affecting downstream signaling pathways that drive proliferation, survival, and metastasis . Studies have shown that monoclonal antibodies that inhibit ADAM17, a key regulator of EGFR ligand availability, can reduce phosphorylation at S1070 along with other sites, correlating with tumor regression or stasis in EGFR-dependent tumor models . This suggests that S1070 phosphorylation contributes to oncogenic EGFR signaling. The inhibitory activity of ADAM17-targeting antibodies correlates with EGFR activity across various tumor types, including head and neck patient-derived xenograft models, indicating potential clinical relevance of these phosphorylation events . In the context of therapeutic resistance, combination antibody treatments that affect receptor levels and phosphorylation patterns (potentially including S1070) have shown superior antitumor activity compared to single agents in certain models like OE21 esophageal cancer and COLO205 colorectal cancer . Such combinations were even able to eradicate tumors in some experimental settings, suggesting that targeting pathways influencing S1070 phosphorylation might help overcome resistance to existing EGFR-targeted therapies . Monitoring S1070 phosphorylation in patient samples before and after treatment could potentially identify biomarkers predicting response or resistance to EGFR-targeted therapies.

What novel methodologies are enhancing the utility of phospho-specific antibodies in EGFR research?

Cutting-edge methodologies are significantly expanding the applications and insights gained from phospho-specific antibodies like Phospho-EGFR (S1070) Antibody in advanced research settings. Single-cell phospho-proteomics approaches using flow cytometry or mass cytometry (CyTOF) with phospho-specific antibodies allow researchers to analyze EGFR phosphorylation heterogeneity within complex cell populations or tumor samples, providing insights that would be masked in bulk analyses . Microfluidic platforms combined with immunofluorescence using phospho-specific antibodies enable high-throughput analysis of phosphorylation dynamics in response to combinatorial stimuli or drug treatments. Proximity ligation assays (PLA) utilizing Phospho-EGFR (S1070) Antibody in combination with antibodies against potential interaction partners reveal spatial and contextual aspects of phosphorylation-dependent protein-protein interactions with exceptional sensitivity. CRISPR-based functional genomic screens coupled with phospho-specific antibody readouts facilitate systematic identification of genes regulating EGFR phosphorylation at specific sites like S1070. Super-resolution microscopy techniques using phospho-specific antibodies provide nanoscale visualization of receptor clustering and signaling complex formation dependent on specific phosphorylation events. Antibody engineering approaches, including site-specific conjugation to fluorophores, quantum dots, or other functional moieties, are expanding applications beyond detection to include targeted drug delivery and theranostics. The integration of these advanced methodologies with computational approaches like machine learning algorithms enables predictive modeling of phosphorylation dynamics and their relationship to cellular outcomes, driving both basic discovery and therapeutic development in EGFR-dependent diseases .

How do available Phospho-EGFR (S1070) antibodies compare in terms of specificity and application versatility?

Commercially available Phospho-EGFR (S1070) antibodies demonstrate varying performance characteristics that researchers should consider when selecting the appropriate reagent for their specific application. While most antibodies are rabbit polyclonal IgGs, differences in immunization strategies, purification methods, and validation protocols result in varying specificity profiles . The following table compares key characteristics of representative Phospho-EGFR (S1070) antibodies:

Specificity validation approaches vary between manufacturers, with some providing more comprehensive validation data than others . Cross-reactivity with other phosphorylated epitopes on EGFR or related receptors is an important consideration, particularly in contexts where multiple ErbB family members are expressed. Application versatility differs significantly, with some antibodies validated only for Western blotting while others may be suitable for additional techniques such as immunoprecipitation, immunofluorescence, or flow cytometry . When comparing antibodies, researchers should evaluate the quality and extent of validation data provided by manufacturers, consider independent validation studies in the literature, and potentially perform their own validation experiments using appropriate controls to ensure the selected antibody meets their specific research requirements .

What are the most common technical challenges when working with Phospho-EGFR (S1070) Antibody and how can they be overcome?

Working with Phospho-EGFR (S1070) Antibody presents several technical challenges that can be systematically addressed through optimized protocols and troubleshooting strategies. One fundamental challenge is preserving phosphorylation during sample preparation; this can be overcome by using ice-cold lysis buffers containing both protease and phosphatase inhibitor cocktails, processing samples rapidly, and avoiding multiple freeze-thaw cycles . Low signal intensity often occurs due to the transient nature of phosphorylation events; optimizing stimulation conditions (ligand concentration, timing) and using positive controls from cells with confirmed S1070 phosphorylation can help establish appropriate detection parameters . Non-specific binding leading to background or multiple bands can be addressed through more stringent washing (increased duration and number of washes), optimization of blocking conditions (5% BSA rather than milk, which contains phosphatases), and titration of primary and secondary antibody concentrations . Inconsistent results between experiments often stem from variations in cell culture conditions or sample processing; standardizing protocols for cell density, serum starvation duration, stimulation parameters, and lysis methods significantly improves reproducibility. Antibody cross-reactivity with other phosphorylated epitopes can be assessed and mitigated through peptide competition assays, using phosphatase-treated controls, and comparing results with other phospho-specific antibodies . For quantitative applications, the limited dynamic range of chemiluminescent detection can be overcome by using fluorescent secondary antibodies and digital imaging systems, which provide more accurate quantification across a broader range of signal intensities.

What are the emerging research areas where Phospho-EGFR (S1070) Antibody will have significant impact?

Phospho-EGFR (S1070) Antibody is poised to make significant contributions to several emerging research areas at the intersection of cell signaling, cancer biology, and precision medicine. In cancer immunotherapy research, understanding how EGFR phosphorylation states, including S1070 phosphorylation, influence tumor-immune interactions and modulate responses to immune checkpoint inhibitors could reveal new combination treatment strategies . The rapidly expanding field of spatial proteomics, employing techniques like imaging mass cytometry and highly multiplexed immunofluorescence, will benefit from phospho-specific antibodies to map signaling activities within the tumor microenvironment with unprecedented resolution. Drug resistance mechanisms in targeted therapies remain a critical challenge; Phospho-EGFR (S1070) Antibody could help identify phosphorylation-dependent resistance pathways and biomarkers predictive of treatment response . In the realm of liquid biopsies, detection of phosphorylated EGFR in circulating tumor cells or exosomes using sensitive antibody-based assays could provide minimally invasive monitoring of treatment responses. Single-cell signaling analysis is revealing striking heterogeneity in cancer cell populations; phospho-specific antibodies enable identification of rare cell subpopulations with distinct signaling states that might drive disease progression or therapeutic resistance. Combination therapies targeting multiple nodes in the EGFR signaling network show promise for overcoming resistance; Phospho-EGFR (S1070) Antibody can help identify synergistic drug combinations by monitoring pathway reactivation mechanisms . Finally, as CRISPR gene editing approaches cancer therapy applications, understanding how genetic alterations affect phosphorylation patterns will be crucial for predicting therapeutic outcomes and designing effective editing strategies.

What developments are needed to enhance our understanding of EGFR S1070 phosphorylation in normal physiology and disease?

Several key developments are needed to deepen our understanding of EGFR S1070 phosphorylation and its significance in both physiological and pathological contexts. First, identification of the specific kinases and phosphatases that directly regulate S1070 phosphorylation is essential; this could be achieved through systematic kinase/phosphatase inhibitor screens, CRISPR knockout libraries, or targeted proteomics approaches . More sensitive and versatile detection tools for S1070 phosphorylation, including antibodies optimized for immunohistochemistry, flow cytometry, and super-resolution microscopy, would enable more comprehensive analysis across diverse experimental and clinical contexts . Development of conformation-specific antibodies that recognize distinct structural states of EGFR associated with S1070 phosphorylation could reveal how this modification affects receptor dynamics. Generation of phospho-mimetic (S1070D/E) and phospho-deficient (S1070A) EGFR knock-in cell lines and animal models would provide definitive systems for studying the functional consequences of this modification in vivo. Comprehensive analysis of S1070 phosphorylation across large patient cohorts, correlated with clinical outcomes and treatment responses, could establish its value as a prognostic or predictive biomarker in EGFR-dependent cancers . Integration of S1070 phosphorylation data with structural biology approaches, including cryo-EM studies of EGFR in different phosphorylation states, might reveal how this modification affects receptor conformation and interactions . Finally, development of computational models incorporating S1070 phosphorylation into the broader EGFR signaling network could generate testable predictions about its role in normal physiology and disease contexts, guiding future experimental approaches and potentially identifying novel therapeutic strategies .