Phospho-ERBB2 (Tyr877) Antibody

What is the biological significance of Tyr877 phosphorylation in ErbB2/HER2?

Tyrosine 877 phosphorylation represents a critical regulatory site within the kinase domain of ErbB2/HER2. This site is homologous to Tyr416 of pp60c-Src and plays a fundamental role in regulating ErbB2 biological activity . Unlike the major autophosphorylation sites (Tyr1248 and Tyr1221/1222) that couple ErbB2 to the Ras-Raf-MAP kinase signal transduction pathway, Tyr877 phosphorylation appears to be involved in more nuanced regulation of receptor function . Given that ErbB2 gene amplification and protein overexpression are detected in approximately 40% of human breast cancers, understanding the regulatory function of this specific phosphorylation site has significant implications for cancer research and therapeutic development .

How does Phospho-ERBB2 (Tyr877) Antibody differ from antibodies targeting other phosphorylation sites?

Phospho-ERBB2 (Tyr877) antibodies specifically detect ErbB2 only when phosphorylated at tyrosine 877, distinguishing them from antibodies targeting other phosphorylation sites such as Thr686, Tyr1112, Tyr1221/1222, or Tyr1248 . This specificity is achieved through careful antibody production methods, including affinity purification on phosphopeptides and removal of non-phosphopeptide-reactive antibodies by chromatography on non-phosphorylated peptides . The significance of this distinction lies in the different functional roles of these phosphorylation sites. For example, while Tyr1112 phosphorylation is associated with c-Cbl binding leading to ErbB2 poly-ubiquitination and degradation, and Tyr1221/1222 and Tyr1248 couple to downstream MAPK signaling, Tyr877 phosphorylation appears to have distinct regulatory functions that can be specifically studied using these antibodies .

What species reactivity can researchers expect with commonly available Phospho-ERBB2 (Tyr877) antibodies?

Current commercially available Phospho-ERBB2 (Tyr877) antibodies demonstrate varying species reactivity profiles as summarized in the following table:

The reactivity predictions for additional species are typically based on 100% sequence homology in the antigen sequence, but researchers should note that actual reactivity may require experimental validation . When selecting an antibody for cross-species applications, researchers should prioritize those with confirmed reactivity in their species of interest or consider preliminary validation experiments.

How should researchers optimize Western blotting protocols for detecting low levels of Tyr877 phosphorylation?

Western blotting for Phospho-ERBB2 (Tyr877) requires meticulous optimization to detect potentially low levels of phosphorylation. Begin with enrichment strategies through immunoprecipitation of total ErbB2/HER2 followed by phospho-specific detection. For direct detection from lysates, load at least 30-50μg of total protein from HER2-expressing cells. The following protocol incorporates critical modifications:

• Use fresh lysates prepared with phosphatase inhibitors (sodium orthovanadate, sodium fluoride, and β-glycerophosphate) to preserve phosphorylation status

• Employ longer transfer times (90-120 minutes) for the high molecular weight ErbB2 protein (~185kDa)

• Start with recommended dilutions (1:500-1:1,000 for Leinco #43075; 1:1000 for Cell Signaling #2241), but prepare optimization series

• Include positive controls (e.g., EGF-stimulated cells known to induce Tyr877 phosphorylation)

• Utilize 7.5% polyacrylamide gels to better resolve the 185kDa band

Signal enhancement with highly sensitive chemiluminescent substrates may be necessary, and overnight primary antibody incubation at 4°C typically yields superior results compared to shorter incubations at room temperature .

What considerations should be taken when designing experiments to study the functional consequences of Tyr877 phosphorylation?

Designing experiments to elucidate the functional significance of Tyr877 phosphorylation requires sophisticated approaches that integrate multiple techniques:

• Site-directed mutagenesis studies: Generate Y877F mutants to prevent phosphorylation and Y877E phosphomimetic mutants to simulate constitutive phosphorylation

• Temporal analysis: Establish time-course studies following ligand stimulation, correlating Tyr877 phosphorylation with specific cellular outcomes

• Pharmacological inhibition: Utilize kinase inhibitors targeting potential upstream kinases (consider src family kinases due to homology with Tyr416)

• Co-immunoprecipitation experiments: Identify binding partners that specifically interact with phosphorylated Tyr877

• Parallel monitoring: Simultaneously assess phosphorylation at Tyr877 and other sites (Tyr1221/1222, Tyr1248) to establish hierarchical phosphorylation relationships

The effectiveness of these approaches depends on careful experimental design with appropriate positive and negative controls. Given that Tyr877 is homologous to the activating phosphorylation site in Src (Tyr416), researchers should monitor both ErbB2 kinase activity and potential cross-talk with Src family kinases following manipulations of Tyr877 phosphorylation status .

How can researchers effectively evaluate antibody specificity for phosphorylated Tyr877 versus non-phosphorylated forms?

Rigorous validation of phospho-specific antibody performance is essential for research integrity. A comprehensive approach includes:

• Phosphatase treatment: Divide lysates from cells with high basal Tyr877 phosphorylation and treat one portion with lambda phosphatase; the signal should disappear in treated samples

• Competing peptide assays: Pre-incubate antibody with excess phosphorylated and non-phosphorylated peptides separately; only the phospho-peptide should abolish detection

• Stimulus-response analysis: Demonstrate increased antibody signal following treatments known to enhance Tyr877 phosphorylation

• Site-directed mutagenesis: Show loss of signal in Y877F mutant-expressing cells

• siRNA knockdown: Confirm disappearance of signal when ErbB2 is depleted

Most commercial Phospho-ERBB2 (Tyr877) antibodies undergo rigorous manufacturing validation. For example, the Leinco #43075 antibody is affinity-purified on phosphopeptide with non-phosphopeptide-reactive antibodies removed by chromatography . Similarly, antibodies from Cell Signaling and Affinity Biosciences show specificity for the phosphorylated form. Nevertheless, researchers should perform their own validation in their specific experimental systems .

What are the most common technical issues when using Phospho-ERBB2 (Tyr877) antibodies and how can they be resolved?

Researchers frequently encounter several technical challenges when working with Phospho-ERBB2 (Tyr877) antibodies:

For immunohistochemistry applications, specialized considerations include antigen retrieval optimization and extended primary antibody incubation (up to 48 hours at 4°C for difficult samples). When troubleshooting Western blotting issues, including positive controls with known Tyr877 phosphorylation (such as growth factor-stimulated cell lines) can help distinguish between technical issues and true biological variation .

How should researchers approach comparative studies between Tyr877 phosphorylation and other ErbB2 phosphorylation sites?

Comparative analysis of multiple ErbB2 phosphorylation sites requires methodological rigor to generate meaningful data:

• Sequential immunoblotting protocol:

  • Start with the phospho-specific antibody expected to give the weakest signal

  • Document results, then strip membranes thoroughly (validate stripping efficiency)

  • Reprobe with the next phospho-specific antibody

  • Conclude with total ErbB2 detection for normalization

• Multicolor fluorescent detection:

  • Use antibodies from different species for simultaneous detection

  • Employ species-specific secondary antibodies with distinct fluorophores

  • Analyze co-localization and relative signal intensities

• Quantification approaches:

  • Always normalize phospho-specific signals to total ErbB2 expression

  • Use ratio calculations (e.g., pTyr877/pTyr1221/1222) to evaluate proportional phosphorylation

  • Apply appropriate statistical analyses for repeated measures

• Temporal considerations:

  • Design time-course experiments to capture potential sequential phosphorylation events

  • Consider shorter intervals (seconds to minutes) for immediate phosphorylation dynamics

  • Include longer time points (hours) to detect sustained signaling changes

When integrating data across multiple phosphorylation sites, researchers should account for potential differences in antibody affinity that may confound direct quantitative comparisons between different phosphorylation sites .

What protocol modifications are necessary for detecting Phospho-ERBB2 (Tyr877) in different experimental contexts?

Optimizing detection protocols across experimental contexts requires systematic adjustments:

For immunofluorescence (IF) applications:

• Use higher antibody concentrations than Western blotting (1:100-1:200 dilution)

• Extend primary antibody incubation to overnight at 4°C

• Include phosphatase inhibitors in all fixation and permeabilization buffers

• Consider acetone fixation for improved phospho-epitope preservation

For immunohistochemistry (IHC) applications:

• Optimize antigen retrieval methods (typically citrate buffer pH 6.0)

• Use amplification systems such as tyramide signal amplification for enhanced sensitivity

• Apply antibody at 1:50-1:100 dilution for paraffin sections

• Include phosphatase inhibitors in all buffers

For immunoprecipitation (IP) applications:

• Validate antibody capacity to recognize native protein conformation

• Use protein A/G beads for rabbit polyclonal antibodies

• Process samples rapidly to minimize dephosphorylation

• Consider cross-linking antibody to beads to prevent heavy chain interference in subsequent analysis

Each application requires independent optimization, with particular attention to fixation methods for morphological techniques and lysis conditions for biochemical approaches. These modifications should be systematically tested and documented to establish reproducible protocols for each experimental system .

How does Tyr877 phosphorylation relate to ErbB2 kinase activity compared to other regulatory phosphorylation sites?

The relationship between Tyr877 phosphorylation and ErbB2 kinase activity presents a complex regulatory picture that differs from other phosphorylation sites. Based on the structural homology with Src's activating phosphorylation site (Tyr416), Tyr877 phosphorylation is believed to play a role in regulating the catalytic activity of ErbB2 . This contrasts with the C-terminal phosphorylation sites (Tyr1221/1222 and Tyr1248), which primarily function as docking sites for adaptor proteins that initiate downstream signaling cascades rather than directly modulating kinase activity .

What are the implications of cross-species differences in ErbB2 Tyr877 phosphorylation research?

Cross-species research on ErbB2 Tyr877 phosphorylation reveals important evolutionary conservation and divergence with significant implications for translational research:

• Sequence conservation: The region surrounding Tyr877 shows high conservation across mammals, with some antibodies recognizing human, mouse, and rat variants . This conservation suggests fundamental importance in receptor function.

• Species-specific signaling dynamics: Despite sequence homology, the regulation and consequences of Tyr877 phosphorylation may differ between species. Mouse models may show different phosphorylation kinetics or upstream regulatory mechanisms compared to human systems.

• Model selection considerations: When selecting animal models for ErbB2 phosphorylation studies, researchers should consider:

  • Verification of antibody cross-reactivity in the specific model system

  • Potential differences in tissue-specific expression patterns

  • Variations in the ErbB receptor family member expression ratios

  • Differences in ligand availability and ligand-induced phosphorylation patterns

• Translational implications: Findings from rodent models require careful validation in human systems before clinical applications. Conserved phosphorylation mechanisms provide stronger translational potential than those showing species variation.

Researchers conducting cross-species studies should include species-specific positive controls and directly compare phosphorylation dynamics between species within the same experimental paradigm to identify potential divergences in regulatory mechanisms .

How can researchers integrate Phospho-ERBB2 (Tyr877) data with broader ErbB signaling network analysis?

Integrating Tyr877 phosphorylation data into broader ErbB network analysis requires sophisticated experimental and computational approaches:

These integrative approaches help contextualize the specific role of Tyr877 phosphorylation within the complex ErbB2 signaling landscape, potentially revealing novel therapeutic vulnerabilities in ErbB2-dependent cancers .