Phospho-ERBB4 (Tyr1284) Antibody

What is Phospho-ERBB4 (Tyr1284) Antibody and what epitope does it recognize?

Phospho-ERBB4 (Tyr1284) antibody is a polyclonal antibody that specifically detects endogenous levels of HER4 only when phosphorylated at Tyrosine 1284. This antibody was generated using synthetic phosphopeptides derived from human HER4 around the phosphorylation site of Tyr1284 (typically within amino acid range 1250-1299) . The antibody is designed with high specificity to bind to the ERBB4 protein exclusively when the tyrosine residue at position 1284 is phosphorylated, making it valuable for studying active signaling states of the receptor . Most commercially available versions are rabbit polyclonal antibodies that have been affinity-purified using epitope-specific phosphopeptides to ensure specificity .

What is the biological significance of ERBB4 phosphorylation at Tyr1284?

Phosphorylation at Tyr1284 represents a critical activation state in ERBB4/HER4 signaling pathways. ERBB4 is a member of the type I receptor protein tyrosine subfamily that includes EGFR, ERBB2, and ERBB3 . When activated by ligands such as neuregulins, heregulin, or NTAK (neural and thymus-derived activator for ErbB kinases), ERBB4 undergoes autophosphorylation at specific tyrosine residues, including Tyr1284 . This phosphorylation event initiates downstream signaling cascades that regulate cellular metabolism, transcription, cell cycle progression, cytoskeletal rearrangement, cell movement, apoptosis, and differentiation . The Tyr1284 phosphorylation site appears to be particularly important in transforming growth factor signals and may play a significant role in cancer development and progression, as ErbB4 levels have been found to be elevated in certain human tumor cell lines .

How does ERBB4 expression vary across different tissue and cell types?

ERBB4 expression demonstrates significant tissue and cell-type specificity. According to research findings, ERBB4 is most predominantly expressed in normal skeletal muscle, heart, pituitary, brain, and cerebellum . In breast cancer research, varying levels of ERBB4 expression have been documented across different cell lines. Higher expression levels are observed in breast tumor cell lines such as T47-D, MDA-MB-453, BT-474, and H3396, which show the highest levels of mRNA . Intermediate expression levels are seen in MCF-7, MDA-MB-330, and MDA-MB-361 cell lines . In contrast, expression of ERBB4 is low or absent in some breast tumor cell lines including MDA-MB-231, MDA-MB-157, MDA-MB-468, and SKBR-3 . This differential expression pattern makes ERBB4 an interesting target for cancer research and potentially for diagnostic applications.

What are the validated research applications for Phospho-ERBB4 (Tyr1284) Antibody?

Phospho-ERBB4 (Tyr1284) antibodies have been validated for multiple research applications with specific recommended dilution ranges for each:

The antibody has been specifically validated using human breast carcinoma samples for immunohistochemistry and HUVEC cells treated with EGF (200ng/ml for 30 minutes) for Western blot applications . For immunofluorescence, validation has been performed using HeLa cells treated with EGF (200nM for 5 minutes) . These validations typically include blocking with phospho-peptides to confirm specificity for the phosphorylated form of the protein .

What is the recommended protocol for detecting phosphorylated ERBB4 in Western blot experiments?

For optimal detection of phosphorylated ERBB4 in Western blot experiments, researchers should follow these methodological steps:

• Cell preparation: Grow cells to approximately 80% confluence and starve overnight in serum-free DMEM/F12 containing 0.1% bovine serum albumin (BSA) .

• Stimulation: Treat cells with appropriate ligands such as heregulin-β1 (HRG-β1) or HB-EGF at 50 ng/ml for 10 minutes at 37°C to induce phosphorylation .

• Cell lysis: Wash cells three times with ice-cold Ca²⁺- and Mg²⁺-free phosphate-buffered saline (PBS) and lyse with ice-cold TGH buffer (1% Triton X-100, 10% glycerol, 20 mM HEPES, pH 7.2, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM Na₃VO₄) .

• Sample incubation: Incubate lysates for 20 minutes on ice with intermittent vortexing to ensure complete protein extraction .

• Sample preparation: Proceed with standard SDS-PAGE sample preparation, including addition of reducing agents and heat denaturation.

• Antibody incubation: Use the Phospho-ERBB4 (Tyr1284) antibody at the recommended dilution (typically 1:500-1:2000) and incubate according to the manufacturer's recommendations.

• Controls: Include both positive controls (e.g., HUVEC cells treated with EGF) and negative controls (untreated cells and/or blocking with phospho-peptide) .

This protocol maximizes the probability of detecting the phosphorylated form of ERBB4 at Tyr1284 while minimizing background and non-specific binding.

How should researchers prepare samples for immunohistochemistry with this antibody?

Effective immunohistochemical detection of phosphorylated ERBB4 requires careful sample preparation:

• Tissue fixation: Fix tissue samples in 10% neutral buffered formalin for 24-48 hours at room temperature.

• Processing and embedding: Process tissues through graded alcohols and xylene before embedding in paraffin.

• Sectioning: Cut paraffin blocks into 4-5 μm thick sections and mount on positively charged slides.

• Antigen retrieval: This is a critical step for phospho-epitopes. Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective for phospho-ERBB4 detection. Heat at 95-100°C for 15-20 minutes.

• Blocking: Block endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 minutes, followed by protein blocking with 5% normal goat serum.

• Antibody application: Apply Phospho-ERBB4 (Tyr1284) antibody at the recommended dilution (typically 1:100-1:300) and incubate overnight at 4°C.

• Detection: Use an appropriate detection system compatible with rabbit primary antibodies.

• Controls: Include positive control tissues known to express phosphorylated ERBB4 (e.g., certain breast carcinomas) and negative controls by either omitting the primary antibody or using competing phospho-peptide to demonstrate specificity .

Preliminary validation studies have demonstrated successful detection in human breast carcinoma samples, which can serve as positive controls for new experimental setups .

How can this antibody be used to study the role of ERBB4 in cancer progression?

Phospho-ERBB4 (Tyr1284) antibody enables researchers to investigate several aspects of ERBB4's role in cancer progression:

• Activation state assessment: The antibody allows for specific detection of the activated form of ERBB4, enabling researchers to determine whether the receptor is in an active signaling state in various cancer types and stages .

• Correlation studies: By examining the relationship between phosphorylated ERBB4 levels and clinical parameters (tumor grade, stage, patient survival), researchers can elucidate the prognostic significance of ERBB4 activation.

• Therapeutic response monitoring: The antibody can be used to assess changes in ERBB4 phosphorylation following treatment with various therapeutic agents, potentially serving as a pharmacodynamic biomarker.

• Pathway analysis: By examining ERBB4 phosphorylation alongside other signaling molecules, researchers can map the signaling networks active in different cancer contexts.

• Isoform-specific studies: When combined with antibodies recognizing specific ERBB4 isoforms, researchers can determine which variants are preferentially activated in different cancer types .

This antibody has particular relevance in breast cancer research, where ERBB4 expression varies significantly between cell lines. For instance, comparing phosphorylation status between high-expressing lines (T47-D, MDA-MB-453, BT-474, H3396) and low-expressing lines (MDA-MB-231, MDA-MB-157, MDA-MB-468, SKBR-3) may yield insights into the role of ERBB4 activation in different breast cancer subtypes .

What methodological approaches can be used to study ErbB4 isoform-specific phosphorylation?

Studying isoform-specific phosphorylation of ERBB4 requires sophisticated methodological approaches:

• Combined antibody approach: Use Phospho-ERBB4 (Tyr1284) antibody in conjunction with isoform-specific antibodies in sequential or dual immunostaining protocols. Antibodies raised against the C-terminus of ERBB4 (such as c-18 and Ab2) can recognize all splice variants of ERBB4 , while isoform-specific antibodies can distinguish between variants.

• Transfection studies: Develop cell lines stably expressing specific human ERBB4 isoforms using expression constructs. Following the methodology described in the literature, MDCK II cells can be transfected with pcDNA3.1 containing human ERBB4 isoform cDNAs, using appropriate transfection reagents . Stable transfectants can be generated after selection with 1 mg/ml G418 for 3-4 weeks, and positive clones confirmed by immunoblotting .

• Stimulation experiments: After establishing isoform-specific expressing cell lines, compare phosphorylation responses to different ligands (e.g., HRG-β1 or HB-EGF at 50 ng/ml) by analyzing tyrosine phosphorylation at the Tyr1284 site .

• Co-immunoprecipitation: Immunoprecipitate with isoform-specific antibodies followed by immunoblotting with Phospho-ERBB4 (Tyr1284) antibody to determine which isoforms undergo phosphorylation under specific conditions.

• Mass spectrometry: For the most comprehensive analysis, perform phospho-proteomics using mass spectrometry to identify all phosphorylation sites on different ERBB4 isoforms and quantify their relative abundance.

These approaches allow researchers to determine whether specific ERBB4 isoforms are preferentially phosphorylated at Tyr1284 in response to different stimuli or in different cellular contexts.

What are the critical parameters for quantitative analysis of ERBB4 phosphorylation?

For reliable quantitative analysis of ERBB4 phosphorylation, researchers should consider these critical parameters:

• Sample preparation consistency: Ensure consistent sample handling, including standardized cell stimulation protocols (e.g., using 50 ng/ml HRG-β1 or HB-EGF for precisely 10 minutes at 37°C) and identical lysis conditions to minimize variability.

• Phosphatase inhibition: Include robust phosphatase inhibitors (e.g., 1 mM Na₃VO₄) in all buffers to prevent artificial dephosphorylation during sample preparation.

• Loading controls: Use appropriate loading controls (total ERBB4, housekeeping proteins) to normalize phospho-ERBB4 signals and account for variations in total protein amount.

• Standard curves: When performing ELISA-based quantification, generate standard curves using recombinant phosphorylated proteins or synthetic phosphopeptides.

• Signal normalization: For Western blot analysis, normalize phospho-ERBB4 signal to total ERBB4 to determine the proportion of receptor that is phosphorylated rather than just the absolute amount of phosphorylated receptor.

• Image analysis: Use appropriate software for densitometric analysis of immunoblots, ensuring the analysis is performed within the linear range of detection.

• Biological replicates: Perform at least three independent biological replicates to account for biological variability and enable statistical analysis.

• Technical controls: Include positive controls (e.g., EGF-stimulated HUVEC cells) and negative controls (competing phospho-peptide) in each experiment to validate assay performance.

Adherence to these parameters ensures that quantitative analyses of ERBB4 phosphorylation are reliable and reproducible across different experimental conditions and between different researchers.

What are common sources of false results when using Phospho-ERBB4 (Tyr1284) Antibody?

Researchers should be aware of several potential sources of false results when working with Phospho-ERBB4 (Tyr1284) antibody:

• False negatives:

  • Inadequate sample preservation: Rapid dephosphorylation can occur if phosphatase inhibitors are insufficient or if samples are not processed quickly enough.

  • Ineffective antigen retrieval: Phospho-epitopes are particularly sensitive to fixation and may require optimized antigen retrieval methods.

  • Antibody degradation: Improper storage or repeated freeze-thaw cycles can reduce antibody efficacy.

  • Low expression levels: Some cell lines (e.g., MDA-MB-231, MDA-MB-157, MDA-MB-468, and SKBR-3) express low or undetectable levels of ERBB4 .

• False positives:

  • Cross-reactivity with other phosphorylated ErbB family members: Despite purification efforts, antibodies may cross-react with similar phosphorylation motifs in related proteins.

  • Non-specific binding: Insufficient blocking or high antibody concentrations can lead to non-specific signals.

  • Autofluorescence or endogenous peroxidase activity: These can be misinterpreted as positive signals in IF or IHC applications.

To mitigate these issues, researchers should:

• Include appropriate positive controls (e.g., EGF-stimulated HUVEC cells or HeLa cells)

• Perform blocking experiments with competing phosphopeptides to confirm signal specificity

• Use cell lines with known ERBB4 expression profiles as references

• Validate findings using multiple detection methods where possible

How can researchers optimize signal detection in low-expression systems?

When working with samples that have low ERBB4 expression or phosphorylation levels, several optimization strategies can enhance signal detection:

• Signal amplification techniques:

  • Use tyramide signal amplification (TSA) systems for immunohistochemistry or immunofluorescence

  • Consider more sensitive ECL substrates for Western blot

  • Employ biotin-streptavidin amplification systems

• Sample enrichment:

  • Perform immunoprecipitation to concentrate ERBB4 protein before Western blot analysis

  • Use phospho-tyrosine antibodies for initial enrichment followed by ERBB4-specific detection

• Stimulation optimization:

  • Determine optimal ligand concentration and stimulation time for maximal phosphorylation

  • Test multiple ligands (HRG-β1, HB-EGF, etc.) as different cell types may respond differently

• Antibody protocol optimization:

  • Extend primary antibody incubation time (e.g., overnight at 4°C)

  • Optimize antibody concentration through careful titration experiments

  • Test different buffer compositions to enhance antibody binding

• Instrumentation settings:

  • For fluorescence applications, optimize exposure settings and use spectral unmixing to reduce background

  • For Western blot, use longer exposure times within the linear range of detection

• Alternative detection methods:

  • Consider using ELISA-based detection which can be more sensitive than Western blot for quantification

  • Explore proximity ligation assay (PLA) for in situ detection of protein-protein interactions involving phosphorylated ERBB4

These optimization strategies can significantly improve the detection of phosphorylated ERBB4 in systems with low expression levels, enabling researchers to study ERBB4 signaling in a wider range of experimental contexts.

What storage and handling practices ensure optimal antibody performance?

To maintain optimal performance of Phospho-ERBB4 (Tyr1284) antibody over time, researchers should adhere to these storage and handling practices:

• Storage temperature: Store the antibody at -20°C for up to 1 year from the date of receipt . Avoid storing at 4°C for extended periods.

• Aliquoting: Upon receipt, prepare small single-use aliquots to minimize freeze-thaw cycles. Each freeze-thaw cycle can reduce antibody activity.

• Thawing procedure: Thaw antibody aliquots on ice or at 4°C, never at room temperature or by applying heat.

• Working dilution preparation: Prepare working dilutions immediately before use and discard any unused diluted antibody.

• Contamination prevention: Use sterile technique when handling the antibody to prevent microbial contamination.

• Transportation: When transporting the antibody between laboratories, ensure it remains frozen using dry ice.

• Buffer compatibility: Avoid introducing incompatible buffers or chemicals that might denature the antibody.

• Documentation: Maintain records of receipt date, aliquoting, and usage to track antibody age and performance over time.

How can Phospho-ERBB4 (Tyr1284) Antibody be used in multiplex analysis systems?

Multiplex analysis systems offer powerful approaches for studying ERBB4 phosphorylation in the context of broader signaling networks:

• Multiplex immunofluorescence:

  • Combine Phospho-ERBB4 (Tyr1284) antibody with antibodies against other signaling molecules

  • Use spectrally distinct fluorophores for each target

  • Employ multispectral imaging systems to separate signals from multiple fluorophores

  • This approach allows visualization of co-localization between phosphorylated ERBB4 and other proteins of interest

• Multiplex Western blotting:

  • Utilize fluorescently labeled secondary antibodies with different emission spectra

  • Perform sequential probing with different primary antibodies

  • This allows detection of multiple proteins or phosphorylation sites on the same membrane

• Mass cytometry (CyTOF):

  • Label Phospho-ERBB4 (Tyr1284) antibody with rare earth metals

  • Combine with other metal-labeled antibodies

  • Analyze cells using mass cytometry for single-cell resolution of multiple parameters

• Antibody arrays:

  • Include Phospho-ERBB4 (Tyr1284) antibody in custom phospho-proteomic arrays

  • Analyze multiple phosphorylation events simultaneously

  • This approach enables high-throughput screening of signaling pathway activation

• Single-cell Western blot:

  • Apply Phospho-ERBB4 (Tyr1284) antibody in microfluidic single-cell Western blot systems

  • Analyze heterogeneity in ERBB4 phosphorylation at the single-cell level

These multiplex approaches allow researchers to place ERBB4 phosphorylation in the broader context of cellular signaling networks, providing more comprehensive insights into its role in normal physiology and disease states.

What are the current limitations in phospho-specific ERBB4 research?

Despite advances in research tools, several limitations persist in phospho-specific ERBB4 research:

• Technical challenges:

  • Phosphorylation states are transient and can be lost during sample processing

  • Current antibodies may not distinguish between closely related phosphorylation sites

  • The dynamic range of detection can be limited for quantitative analyses

• Biological complexities:

  • ERBB4 has multiple isoforms with potentially different phosphorylation patterns

  • The functional significance of specific phosphorylation sites is not fully characterized

  • Cross-talk with other signaling pathways complicates interpretation of results

• Methodological gaps:

  • Limited availability of standardized positive controls across different experimental systems

  • Variability in antibody performance between different lots and manufacturers

  • Challenges in reproducing the native cellular environment in in vitro studies

• Translation to clinical applications:

  • Difficulty preserving phosphorylation status in clinical samples

  • Variability in tissue processing protocols affecting phospho-epitope detection

  • Limited correlation data between phosphorylation status and clinical outcomes

These limitations highlight the need for continued development of more specific and sensitive tools for detecting and quantifying ERBB4 phosphorylation, as well as standardized protocols for sample handling and analysis.

What emerging technologies might improve phospho-ERBB4 detection and analysis?

Several emerging technologies show promise for advancing phospho-ERBB4 detection and analysis:

• Nanobody and aptamer-based detection:

  • Development of smaller binding molecules with potentially higher specificity for phospho-epitopes

  • May offer improved tissue penetration and reduced background compared to traditional antibodies

• CRISPR-based reporters:

  • Generation of cell lines with endogenous ERBB4 tagged with fluorescent reporters that change localization upon phosphorylation

  • Allows real-time monitoring of phosphorylation events in living cells

• Advanced mass spectrometry:

  • Improved sensitivity for detecting and quantifying phosphorylation at specific sites

  • Ability to identify novel phosphorylation sites and their dynamics

• Digital spatial profiling:

  • Combined with phospho-specific antibodies to map phosphorylation patterns across tissue sections with spatial resolution

  • Links phosphorylation status to tissue architecture and cellular microenvironment

• Artificial intelligence in image analysis:

  • Machine learning algorithms for automated quantification of phosphorylation signals

  • Reduction in subjective interpretation of immunohistochemistry or immunofluorescence data

These technologies may overcome current limitations in studying ERBB4 phosphorylation and provide more comprehensive insights into its role in normal physiology and disease states.

How might phospho-ERBB4 research translate to clinical applications?

Research on phosphorylated ERBB4 has several potential clinical applications:

• Biomarker development:

  • Phosphorylated ERBB4 could serve as a biomarker for cancer diagnosis, prognosis, or prediction of treatment response

  • Different phosphorylation patterns might distinguish between cancer subtypes

• Therapeutic target identification:

  • Understanding the role of specific phosphorylation sites could reveal new therapeutic targets

  • Inhibitors targeting specific downstream pathways activated by phosphorylated ERBB4

• Companion diagnostics:

  • Phospho-ERBB4 detection could guide the use of targeted therapies that modulate ErbB family signaling

  • Could help select patients most likely to benefit from specific treatments

• Resistance mechanisms:

  • Changes in ERBB4 phosphorylation patterns might indicate development of resistance to current therapies

  • Could guide sequential or combination treatment strategies

• Monitoring treatment response:

  • Changes in phospho-ERBB4 levels during treatment could serve as pharmacodynamic markers

  • Could provide early indication of treatment efficacy before clinical response is evident