Phospho-EGFR (S1071) Antibody

What is the Phospho-EGFR (S1071) Antibody and what epitope does it recognize?

Phospho-EGFR (S1071) Antibody is a polyclonal antibody specifically designed to recognize the Epidermal Growth Factor Receptor (EGFR) protein only when phosphorylated at serine residue 1071. The antibody is typically generated using synthesized peptides derived from human EGFR encompassing the phosphorylation site of S1071 as immunogens . The specificity for the phosphorylated form allows researchers to distinguish the activated state of EGFR at this particular residue from the non-phosphorylated form, enabling detailed signaling studies.

How does Phospho-EGFR (S1071) differ from other phospho-specific EGFR antibodies?

Phospho-EGFR (S1071) antibody specifically targets serine phosphorylation, while many commonly used phospho-EGFR antibodies target tyrosine residues such as Y1068, Y1086/Y1110, or Y1172 . This distinction is important because:

• Serine/threonine phosphorylation often regulates receptor desensitization and internalization, whereas tyrosine phosphorylation typically activates downstream signaling cascades .

• Different kinases are responsible for these modifications - serine/threonine kinases versus tyrosine kinases - representing distinct regulatory mechanisms.

• The functional outcomes of S1071 phosphorylation may differ significantly from those of tyrosine phosphorylation sites.

This table summarizes key differences between common phospho-EGFR antibodies:

What are the recommended experimental applications for Phospho-EGFR (S1071) Antibody?

Based on validation data, Phospho-EGFR (S1071) Antibody is primarily recommended for:

• Western Blotting (WB): Optimal dilution range 1:500-1:2000

  • Can detect bands at approximately 134-180 kDa depending on post-translational modifications

• ELISA: Recommended dilution of 1:5000

  • Useful for quantitative assessment of phosphorylation levels

While some vendors may suggest other applications, these two have the most consistent validation data across manufacturers. For specialized applications like immunohistochemistry (IHC) or immunofluorescence (IF), additional validation may be necessary in your specific experimental system.

What are the optimal sample preparation methods for detecting Phospho-EGFR (S1071)?

For effective detection of Phospho-EGFR (S1071), consider these critical sample preparation guidelines:

• Phosphatase inhibitors: Always include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate) in lysis buffers to preserve phosphorylation status .

• Lysis conditions: Use RIPA or similar buffers supplemented with protease inhibitors. For membrane proteins like EGFR, addition of 0.1% SDS can improve extraction efficiency .

• Sample handling: Process samples rapidly at 4°C to minimize dephosphorylation. Flash freezing in liquid nitrogen is recommended if immediate processing is not possible.

• Positive controls: EGF-stimulated A431 cells (human epithelial carcinoma) serve as excellent positive controls, showing robust phosphorylation at S1071 after treatment with 100 ng/mL recombinant human EGF for 5-10 minutes .

• Reduction and denaturation: Complete denaturation is critical; use reducing conditions (DTT or β-mercaptoethanol) and heat samples at 95°C for 5 minutes.

How should I design experiments to study the kinetics of EGFR S1071 phosphorylation?

To effectively study the kinetics of EGFR S1071 phosphorylation:

• Time course experiment design:

  • Establish baseline (0 min) phosphorylation levels

  • Stimulate cells with appropriate ligand (typically EGF at 100 ng/mL)

  • Collect samples at multiple timepoints (30 sec, 2 min, 5 min, 10 min, 30 min, 1 hr, 2 hr)

  • Process all samples simultaneously to minimize technical variation

• Quantification approach:

  • Always normalize phospho-signal to total EGFR expression levels using a non-phospho-specific EGFR antibody on stripped membranes

  • Use densitometry software for accurate quantification

  • Present data as fold-change in phosphorylation relative to baseline

• Controls to include:

  • Unstimulated controls at each timepoint to account for basal changes

  • Phosphatase treatment controls to confirm signal specificity

  • EGFR inhibitor controls (e.g., erlotinib) to demonstrate signal regulation

Research has shown that different phosphorylation sites on EGFR demonstrate distinct temporal patterns, with S1071 potentially showing different kinetics compared to tyrosine sites like Y1068 or Y1086 .

How should I interpret discrepancies between phospho-EGFR (S1071) levels and other EGFR phosphorylation sites?

Discrepancies between phosphorylation levels at different EGFR sites are common and biologically significant. When analyzing such differences:

• Different regulatory mechanisms: Serine phosphorylation (including S1071) is often mediated by downstream kinases rather than receptor autophosphorylation. For example, studies have shown that while EGF stimulation induces rapid Y1068 phosphorylation, S1071 phosphorylation may follow different kinetics and might be regulated by distinct pathways .

• Functional implications: Tyrosine phosphorylation sites (e.g., Y1068, Y1086) primarily mediate downstream signaling through recruitment of adaptor proteins like GRB2, while serine phosphorylation often regulates receptor trafficking, desensitization, or cross-talk with other pathways .

• Cell type-specific patterns: Different cell types may exhibit distinct phosphorylation patterns. For example, research on EGFR phosphorylation in lung cancer cells (A549) versus breast cancer cells (MDA-MB-231) showed distinct patterns even with the same stimulus .

• Interpretative approach: When observing discrepancies, consider:

  • Is the difference stimulus-specific?

  • Does inhibition of specific pathways differentially affect these sites?

  • Are these differences consistent across cell types or unique to your model?

What are common sources of false positives/negatives when using Phospho-EGFR (S1071) antibodies?

Several factors can contribute to false results when using phospho-specific antibodies:

Potential sources of false positives:

• Cross-reactivity with similar phosphorylation motifs on other proteins

• Insufficient blocking during Western blot procedures

• Sample overloading causing non-specific binding

• Inadequate washing steps in immunoassays

Potential sources of false negatives:

• Rapid dephosphorylation during sample preparation (insufficient phosphatase inhibitors)

• Epitope masking due to protein-protein interactions

• Suboptimal antibody concentration

• Incomplete protein transfer during Western blotting for large proteins like EGFR

Verification strategies:

• Phosphatase treatment controls - sample treatment with lambda phosphatase should abolish signal

• Peptide competition assays - pre-incubation with phosphopeptide should block specific binding

• siRNA knockdown of EGFR - should reduce or eliminate specific signal

• Comparison with other well-characterized phospho-specific antibodies using the same samples

How can Phospho-EGFR (S1071) Antibody be used to study receptor trafficking and internalization?

Phosphorylation at serine residues like S1071 has been implicated in EGFR internalization and trafficking. To study these processes:

• Subcellular fractionation approach:

  • Separate cellular compartments (membrane, cytosolic, endosomal fractions)

  • Analyze the distribution of phospho-EGFR (S1071) versus total EGFR

  • Compare with markers for different cellular compartments (e.g., Na+/K+ ATPase for plasma membrane, EEA1 for early endosomes)

• Imaging techniques:

  • Combined immunofluorescence using phospho-EGFR (S1071) antibody with compartment markers

  • Live-cell imaging using fluorescently tagged EGFR combined with phospho-specific antibody staining after fixation

  • Super-resolution microscopy for detailed localization studies

• Receptor trafficking inhibitors:

  • Use specific inhibitors of different trafficking pathways (e.g., dynamin inhibitors, clathrin inhibitors)

  • Measure effects on S1071 phosphorylation compared to other EGFR phosphorylation sites

  • Compare with receptor downregulation measurement using surface biotinylation assays

Research has shown that antibody combinations targeting different EGFR domains can reduce surface receptor levels by up to 80%, suggesting complex regulation of receptor internalization . The relationship between S1071 phosphorylation and this process remains an active area of investigation.

What is the role of Phospho-EGFR (S1071) in cancer resistance mechanisms to EGFR-targeted therapies?

EGFR-targeted therapies, including tyrosine kinase inhibitors like erlotinib, are important cancer treatments, but resistance frequently develops. Investigating the role of S1071 phosphorylation in this context:

• Resistance model systems:

  • Compare S1071 phosphorylation levels between sensitive and resistant cell lines

  • Develop acquired resistance models through long-term drug exposure

  • Use patient-derived xenografts from responders versus non-responders

• Signaling bypass mechanisms:

  • Investigate whether S1071 phosphorylation persists despite tyrosine kinase inhibition

  • Determine which upstream kinases maintain S1071 phosphorylation during drug treatment

  • Assess whether combined inhibition of these kinases restores drug sensitivity

• Methodological approaches:

  • Phosphoproteomics to comprehensively map changes in EGFR phosphorylation patterns during resistance development

  • Site-directed mutagenesis (S1071A) to determine functional significance

  • In vivo models to validate findings from cell culture systems

Research has shown that while EGFR tyrosine phosphorylation sites like Y1092, Y1110, and Y1172 correlate with sensitivity to erlotinib , the role of serine phosphorylation sites including S1071 in modulating drug response represents an understudied area that could reveal novel resistance mechanisms.

How do different EGFR mutations affect phosphorylation at S1071 compared to tyrosine phosphorylation sites?

EGFR mutations, particularly in lung cancer, significantly impact receptor function and drug sensitivity. Understanding their effect on S1071 phosphorylation:

• Comparative analysis across mutation types:

  • Common activating mutations (exon 19 deletions, L858R)

  • Resistance mutations (T790M, C797S)

  • Uncommon mutations with variable clinical responses

• Experimental approach:

  • Isogenic cell lines expressing wild-type versus mutant EGFR

  • Analysis of basal and ligand-induced phosphorylation patterns

  • Temporal dynamics of phosphorylation/dephosphorylation at different sites

• Structural considerations:

  • How mutations affect the conformation of the kinase domain

  • Impact on accessibility of S1071 to kinases and phosphatases

  • Potential for altered protein-protein interactions affecting serine phosphorylation

How can mass spectrometry complement antibody-based detection of Phospho-EGFR (S1071)?

Mass spectrometry (MS) provides powerful complementary approaches to antibody-based detection:

• Unbiased phosphorylation site mapping:

  • MS can identify novel and known phosphorylation sites simultaneously

  • Enables discovery of previously uncharacterized sites that may function together with S1071

  • Can detect multiple modifications on the same peptide to reveal combinatorial regulation

• Quantitative assessment advantages:

  • Label-free quantitation allows precise measurement of phosphorylation stoichiometry

  • Multiple reaction monitoring (MRM) provides highly sensitive targeted quantification

  • SILAC or TMT labeling enables multiplexed comparison across conditions

• Methodological workflow:

  • EGFR immunoprecipitation followed by tryptic digestion

  • Phosphopeptide enrichment (TiO2, IMAC, or phospho-specific antibodies)

  • High-resolution LC-MS/MS analysis

  • Database searching with phosphorylation as variable modification

Studies utilizing mass spectrometry have identified 30 EGFR phosphorylation sites, including 12 serine phosphorylation sites, providing a comprehensive map of receptor modification that extends beyond what can be studied with available antibodies . These approaches have revealed that five sites (pT693, pY1092, pY1110, pY1172 and pY1197) are inhibited by erlotinib in a concentration-dependent manner .

What is the interplay between S1071 phosphorylation and other post-translational modifications on EGFR?

EGFR undergoes multiple types of post-translational modifications that may interact with S1071 phosphorylation:

• Cross-talk with other phosphorylation sites:

  • Hierarchical phosphorylation events where one site influences modification of another

  • Competitive phosphorylation where different kinases target overlapping regions

  • Combinatorial effects where specific patterns of multi-site phosphorylation create unique signaling outputs

• Interaction with non-phosphorylation modifications:

  • Ubiquitination - potentially influenced by serine phosphorylation status

  • Glycosylation - may affect accessibility of kinases to specific residues

  • Acetylation - emerging modification with potential regulatory functions

• Experimental approaches:

  • Site-directed mutagenesis to create phosphomimetic (S1071D/E) or phospho-dead (S1071A) mutants

  • Kinase inhibitor profiling to identify relevant upstream enzymes

  • Proteomics analysis of interactomes specific to phosphorylation status

• Methodological considerations:

  • Use of appropriate phosphatase inhibitors to preserve physiological modification patterns

  • Analysis of intact proteins to maintain modification relationships

  • Consideration of spatio-temporal regulation of different modifications

Research has shown that AIB1 knockdown affects both tyrosine and serine/threonine phosphorylation of EGFR, suggesting complex regulatory networks governing receptor modification . Understanding these relationships is critical for developing more effective therapeutic strategies.

How does phosphorylation at S1071 compare between different isoforms and family members of EGFR?

EGFR (ErbB1) belongs to a family of receptors including ErbB2, ErbB3, and ErbB4, with significant implications for comparing phosphorylation patterns:

• Comparative analysis across ErbB family members:

  • Sequence alignment to identify equivalent or distinct phosphorylation sites

  • Conservation of S1071 or analogous sites across the family

  • Distinct regulatory mechanisms potentially affecting these sites

• EGFR isoform considerations:

  • Alternative splicing produces EGFR variants with potentially different regulation

  • Isoform 2 may act as an antagonist of EGF action , potentially with altered phosphorylation patterns

  • Expression pattern differences (e.g., isoform 2 expressed in ovarian cancers )

• Experimental approaches:

  • Isoform-specific detection using selective antibodies

  • Recombinant expression of different family members for comparative analysis

  • Receptor co-expression studies to determine heterodimer effects on phosphorylation

• Functional implications:

  • How phosphorylation patterns influence homo- versus heterodimerization

  • Differential sensitivity to therapeutic antibodies or small molecule inhibitors

  • Potential for isoform-specific targeting strategies

Research indicates that EGFR isoforms may have distinct functions, with isoform 2 potentially acting as an antagonist of EGF action . Understanding the phosphorylation patterns across these variants could reveal important regulatory mechanisms and therapeutic opportunities.

What are emerging technologies for sensitive detection of low-abundance Phospho-EGFR (S1071)?

Recent technological advances have significantly improved detection of low-abundance phosphorylation events:

• Enhanced sensitivity methods:

  • Proximity ligation assay (PLA) - allows visualization of specific phosphorylation events in situ with single-molecule sensitivity

  • Single-molecule pull-down (SiMPull) - combines immunoprecipitation with single-molecule fluorescence detection

  • Nanoscale antibody arrays - miniaturized immunoassays with femtomolar sensitivity

• Signal amplification approaches:

  • Tyramide signal amplification (TSA) - enzymatic deposition of fluorescent tyramide for significantly enhanced signal

  • Rolling circle amplification (RCA) - DNA amplification linked to antibody binding events

  • Quantum dot-conjugated secondary antibodies - brighter and more stable than conventional fluorophores

• Novel sample preparation strategies:

  • Laser capture microdissection to isolate specific cell populations from heterogeneous tissues

  • Digital protein analysis through single-cell western blotting

  • Selective phosphopeptide enrichment using titanium dioxide or immobilized metal affinity chromatography

Research on combination antibody treatments has demonstrated the ability to downregulate EGFR surface expression by up to 80% , highlighting the need for increasingly sensitive detection methods to capture the remaining receptor population and its phosphorylation status.

How can phospho-flow cytometry be optimized for Phospho-EGFR (S1071) detection in heterogeneous samples?

Phospho-flow cytometry offers unique advantages for analyzing phosphorylation events in mixed cell populations:

• Sample preparation optimization:

  • Critical fixation timing to capture transient phosphorylation events

  • Permeabilization protocol selection based on epitope accessibility

  • Buffer compositions that preserve phosphorylation while enabling antibody penetration

• Antibody validation for flow cytometry:

  • Confirmation of specificity in positive control systems (e.g., EGF-stimulated A431 cells)

  • Titration to determine optimal concentration for signal-to-noise ratio

  • Testing with phosphatase-treated controls to confirm phospho-specificity

• Multi-parameter analysis strategies:

  • Combined surface marker and phospho-epitope detection

  • Inclusion of total EGFR measurement for normalization

  • Integration with cell cycle analysis or viability markers

• Data analysis approaches:

  • Phosphorylation-specific gating strategies

  • Population-level versus single-cell analysis

  • Visualization tools for complex phosphorylation relationships

While Phospho-EGFR (S1071) antibodies have not been extensively validated for flow cytometry, approaches similar to those used for phospho-EGFR (Y1068) detection in flow cytometry could be adapted, with appropriate optimization and validation steps.

What is the potential for Phospho-EGFR (S1071) as a biomarker in cancer diagnosis or treatment selection?

Phosphorylation status of EGFR has significant potential as a biomarker in cancer:

• Diagnostic applications:

  • Differential phosphorylation patterns between normal and malignant tissues

  • Correlation with specific cancer subtypes or stages

  • Potential for non-invasive detection in liquid biopsies

• Predictive biomarker potential:

  • Association with response to EGFR-targeted therapies

  • Indication of specific resistance mechanisms

  • Guidance for combination therapy approaches

• Methodological considerations for clinical implementation:

  • Sample collection and preservation protocols to maintain phosphorylation status

  • Standardization of detection methods across clinical laboratories

  • Development of quantitative cutoff values for clinical decision-making

• Evidence from existing research:

  • Studies have identified three sites (pY1092, pY1110, pY1172) correlating with activating mutations while three sites (pY1110, pY1172, pY1197) correlated with erlotinib sensitivity

  • S1071 phosphorylation status could potentially provide complementary information to these established markers

The relationship between S1071 phosphorylation and clinical outcomes remains an important area for investigation, with potential significance for patient stratification and treatment selection strategies.

How does the phosphorylation status of EGFR at S1071 compare between different cancer types and stages?

Understanding the pattern of S1071 phosphorylation across cancer types could provide valuable insights:

• Comparative analysis across cancer types:

  • Epithelial cancers with known EGFR involvement (lung, colorectal, head and neck)

  • Comparison with cancers where EGFR plays a variable role (breast, pancreatic)

  • Correlation with EGFR expression levels and mutation status

• Progression analysis:

  • Changes in phosphorylation patterns during tumor development

  • Association with invasive or metastatic phenotypes

  • Comparison between primary tumors and metastatic lesions

• Methodological approaches:

  • Tissue microarray analysis with phospho-specific immunohistochemistry

  • Quantitative image analysis for standardized scoring

  • Complementary proteomic profiling of selected samples

Research has demonstrated that EGFR phosphorylation patterns differ between cancer types, with studies showing distinct patterns between lung cancer cells (A549), breast cancer cells (MDA-MB-231), and pancreatic cancer cells (PANC-1) . These differences may reflect tissue-specific signaling networks and could influence therapeutic vulnerabilities.

How can computational modeling integrate Phospho-EGFR (S1071) data to understand system-level regulation?

Systems biology approaches can provide valuable insights into complex EGFR signaling networks:

• Kinetic modeling approaches:

  • Ordinary differential equation (ODE) models of EGFR phosphorylation/dephosphorylation

  • Parameter estimation using time-course experimental data

  • Sensitivity analysis to identify critical regulatory nodes

• Network-based analysis:

  • Integration of phospho-EGFR (S1071) data with larger signaling networks

  • Identification of feedback and feedforward loops involving this site

  • Prediction of system-level responses to perturbations

• Multi-scale modeling considerations:

  • Linking molecular events (phosphorylation) to cellular phenotypes

  • Integration of spatial aspects of receptor trafficking

  • Incorporation of tissue-level heterogeneity

• Data requirements and experimental design:

  • Time-resolved measurements across multiple conditions

  • Quantitative data for model training and validation

  • Targeted perturbation experiments to test model predictions

Research has shown that EGFR activates at least four major downstream signaling cascades: RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules . Integrating S1071 phosphorylation data into these signaling networks could reveal novel regulatory mechanisms and therapeutic opportunities.

What are the best experimental designs for studying the dynamics of S1071 phosphorylation in the context of feedback regulation?

EGFR signaling involves complex feedback mechanisms that influence phosphorylation dynamics:

• Temporal sampling strategies:

  • High-density early timepoints (seconds to minutes) to capture rapid phosphorylation events

  • Extended timepoints (hours to days) to observe adaptation and feedback effects

  • Synchronized cell populations to reduce heterogeneity

• Perturbation approaches:

  • Dose-response studies with varying EGF concentrations

  • Pulses versus sustained stimulation paradigms

  • Inhibitor time-course studies with pathway-specific inhibitors

• Readout technologies:

  • Multiplexed detection of multiple phosphorylation sites simultaneously

  • Live-cell reporters for real-time monitoring where feasible

  • Single-cell approaches to capture population heterogeneity

• Experimental considerations for feedback analysis:

  • Combined inhibition of receptor and downstream pathways

  • Chemical-genetic approaches for rapid and specific kinase inhibition

  • Protein synthesis inhibitors to distinguish between immediate and delayed feedback mechanisms