ERBB4 Antibody

What is ERBB4 and what are its main characteristics?

ERBB4, also known as HER4 (human epidermal growth factor receptor 4), is a type I membrane glycoprotein belonging to the ErbB family of tyrosine kinase receptors. It has a calculated molecular weight of 147 kDa but is typically observed at approximately 180 kDa and 147 kDa in western blots . ERBB4 contains multiple functional domains, including a ligand-binding extracellular region and a tyrosine kinase domain .

ERBB4 is activated by several ligands including neuregulins (NRGs), heparin-binding EGF-like growth factor (HB-EGF), betacellulin, and neuregulin transfer activating factor (NTAK) . Upon activation, ERBB4 can form homodimers or heterodimers with other ErbB family members, particularly ErbB2, though heterodimerization with ErbB1 and ErbB3 has also been documented . These interactions are crucial for understanding antibody binding and specificity in research applications.

What tissues and cell types express ERBB4?

ERBB4 is expressed in several normal tissues and cell types, which is important for researchers to consider when selecting appropriate experimental controls. ERBB4 is predominantly found in:

• Normal skeletal muscle

• Heart tissue

• Pituitary gland

• Brain tissues (including hippocampal neurons)

• Several breast carcinoma cell lines

In research settings, MCF-7 human breast cancer cells are frequently used as a positive control for ERBB4 expression, as confirmed by multiple studies . Western blot analyses have consistently shown that MCF-7 cells express detectable levels of ERBB4, while ErbB4 knockout MCF-7 cell lines serve as excellent negative controls . Other recommended positive controls include T47D human breast cancer cells, mouse brain tissue, and rat brain tissue .

How do I validate the specificity of an ERBB4 antibody?

Validating antibody specificity is critical for obtaining reliable research results. For ERBB4 antibodies, a multi-step validation approach is recommended:

• Knockout validation: Compare parental cell lines (e.g., MCF-7) with ERBB4 knockout versions of the same cell line. A specific ERBB4 antibody should detect a band at approximately 180 kDa in the parental line but show no reactivity in the knockout line .

• Western blot analysis: Confirm the detection of bands at the expected molecular weights (approximately 180 kDa and/or 147 kDa) in known ERBB4-expressing tissues such as brain tissue or MCF-7 cells .

• Cross-reactivity testing: Test the antibody against multiple species if cross-reactivity is claimed. Published ERBB4 antibodies have demonstrated reactivity with human, mouse, and rat samples .

• Loading controls: Always include appropriate loading controls (e.g., GAPDH) to ensure equal protein loading when comparing samples .

• Immunoprecipitation validation: Perform immunoprecipitation followed by western blotting with a different ERBB4 antibody to confirm specificity .

What are the optimal conditions for Western blotting with ERBB4 antibodies?

Successful western blotting for ERBB4 requires attention to several technical parameters:

For optimal results, it's recommended to titrate the antibody concentration for each specific application and sample type . Additionally, ensure complete transfer of high molecular weight proteins by adjusting transfer time and conditions appropriately.

How should I optimize immunohistochemistry protocols for ERBB4 detection?

Immunohistochemistry (IHC) detection of ERBB4 requires specific optimization steps:

• Antigen retrieval: Two methods have proven effective:

  • TE buffer at pH 9.0 (primary recommendation)

  • Citrate buffer at pH 6.0 (alternative method)

• Antibody dilution: For IHC applications, a dilution range of 1:50-1:500 is typically recommended, though this should be optimized for each specific antibody and tissue type .

• Positive tissue controls: Mouse lung tissue and human kidney tissue have been validated as positive controls for IHC applications .

• Signal amplification: For tissues with lower ERBB4 expression, consider using amplification systems to enhance signal detection.

• Counterstaining: Use appropriate counterstains that don't interfere with the ERBB4 signal detection.

Research has successfully used these approaches to detect differential ERBB4 expression in various tissues, including increased ERBB4 immunoreactivity in apoptotic hippocampal pyramidal neurons in Alzheimer's disease patients compared to healthy controls .

What considerations are important for immunoprecipitation of ERBB4?

Immunoprecipitation (IP) is a valuable technique for studying ERBB4 interactions and modifications:

• Antibody selection: For phosphorylation studies, a mixture of anti-phosphotyrosine antibodies can be used to immunoprecipitate the phosphorylated form of ERBB4 .

• Cell stimulation: Stimulation with Heregulinβ1 (HRGβ1) for approximately 15 minutes can enhance ERBB4 phosphorylation, although some studies have observed tyrosine phosphorylation of ERBB4 even without ligand stimulation .

• Lysis conditions: Use lysis buffers that preserve protein-protein interactions and post-translational modifications. For phosphorylation studies, include phosphatase inhibitors in all buffers.

• Confirmation: Following IP, confirm the presence of ERBB4 by western blotting using a different ERBB4 antibody than used for the IP to avoid detecting the heavy chain of the IP antibody .

• Downstream applications: The immunoprecipitated ERBB4 can be used for subsequent mass spectrometry analysis to identify phosphorylation sites or interacting proteins .

How can I detect and distinguish between different ERBB4 isoforms?

ERBB4 exists in various isoforms that differ in their juxtamembrane (JM) region and cytoplasmic domain. These isoforms have different biological functions and can influence experimental outcomes:

• JM isoforms: The cleavable JM-a isoform is associated with estrogen receptor-α expression and high histologic grade of differentiation in breast cancer . Specific antibodies or PCR primers can be designed to distinguish between JM isoforms.

• Cytoplasmic isoforms: Different cytoplasmic isoforms (CYT-1 and CYT-2) have distinct signaling capabilities. CYT-1 contains a PI3K binding site absent in CYT-2.

• Localization-based distinction: Cytosolic localization of ERBB4 has been associated with better breast cancer prognosis, while nuclear localization correlates with worse prognosis . Subcellular fractionation followed by western blotting can help distinguish these populations.

• RT-PCR analysis: Real-time RT-PCR using isoform-specific primers is an effective method to quantify the expression of different ERBB4 isoforms in research samples .

• Antibody selection: When studying specific isoforms, select antibodies whose epitopes are present in the isoform of interest. The epitope location can significantly affect experimental results.

How can I study ERBB4 phosphorylation sites and their signaling implications?

ERBB4 phosphorylation is a key regulatory mechanism that influences its signaling activities. Tandem mass spectrometry has identified 19 sites of tyrosine phosphorylation on ERBB4 . To study these sites:

• Site-specific phospho-antibodies: When available, use antibodies that recognize specific phosphorylated residues of ERBB4.

• Mass spectrometry approach: For comprehensive phosphorylation analysis:

  • Transfect cells with ERBB4 expression constructs

  • Stimulate with ligands such as Heregulinβ1

  • Immunoprecipitate ERBB4 using anti-phosphotyrosine antibodies

  • Separate by SDS-PAGE and visualize by Coomassie staining

  • Perform in-gel digestion followed by tandem mass spectrometry

• Protein microarrays: These can be used to quantify biophysical interactions between phosphorylated sites and various SH2 and PTB domains, providing insights into downstream signaling pathways .

• Functional consequences: Research has shown that ERBB4 can recruit and activate STAT1, a finding revealed through systematic investigation of its phosphorylation sites . Similar approaches can identify other novel interactions.

• Mutation studies: To assess the importance of specific phosphorylation sites, consider creating phospho-mimetic (Y to E) or phospho-deficient (Y to F) mutations at sites of interest.

How do ERBB4 antibodies perform in three-dimensional culture systems?

Three-dimensional (3D) culture systems more accurately reflect the in vivo environment than traditional 2D cultures, particularly for studying cancer biology:

• Antibody penetration: In 3D culture systems, ensure adequate antibody penetration by optimizing incubation times and potentially using permeabilization agents.

• Blocking: More extensive blocking may be required to reduce background in 3D cultures compared to 2D systems.

• Application in research: Anti-ERBB4 monoclonal antibodies (such as clone P6-1) have been successfully used to examine the effect of ERBB4 inhibition on breast cancer cell proliferation in extracellular matrix 3D cultures .

• Visualization techniques: Confocal microscopy is often necessary to properly visualize ERBB4 staining in 3D cultures, allowing for optical sectioning through the sample.

• Functional studies: Neutralizing ERBB4 antibodies can be used in 3D cultures to study the role of neuregulin-dependent activation of ERBB4 in a more physiologically relevant context .

How does ERBB4 expression differ between cancer types and what are the implications for antibody-based research?

ERBB4 shows distinct expression patterns and functions across different cancer types, which has important implications for antibody-based research:

When designing experiments, researchers should consider cancer-specific expression patterns and use appropriate positive and negative controls relevant to the cancer type being studied.

What is the role of ERBB4 in neurodegenerative diseases and how can antibodies help study this?

ERBB4 has been implicated in several neurodegenerative conditions, offering opportunities for antibody-based research approaches:

• Alzheimer's Disease (AD): Studies have found significantly higher ERBB4 immunoreactivity in apoptotic hippocampal pyramidal neurons in AD patients compared to healthy controls . ERBB4 is also found at high levels surrounding the neuritic plaques characteristic of AD.

• Parkinson's Disease (PD): Elevated ERBB4 expression has been observed in midbrain tissue sections of PD patients compared with healthy controls .

• Antibody applications:

  • Immunohistochemistry to map expression changes in diseased versus healthy tissue

  • Co-localization studies to examine ERBB4 proximity to disease markers (e.g., amyloid plaques)

  • Western blotting of brain tissue lysates to quantify expression level changes

• Challenges: Brain tissue may require special processing for optimal antibody performance, including extended fixation times and specific antigen retrieval methods.

• Potential therapeutic relevance: Understanding ERBB4's role in neurodegeneration could reveal whether it represents a protective response or contributes to disease progression, information that could guide therapeutic development.

How can neutralizing ERBB4 antibodies be used in functional studies?

Neutralizing antibodies that block ERBB4 activation represent valuable tools for studying its function in various contexts:

• Mechanism of action: Neutralizing anti-ERBB4 monoclonal antibodies (such as clone P6-1) can suppress neuregulin-dependent activation of ERBB4 . These antibodies typically target the ligand-binding domain to prevent receptor activation.

• Applications in cancer research: Neutralizing antibodies have been used to examine the effect of ERBB4 inhibition on breast cancer cell proliferation in the extracellular matrix .

• Signaling pathway investigation: By selectively blocking ERBB4 activation, researchers can dissect the specific contribution of ERBB4 to downstream signaling cascades.

• Experimental design considerations:

  • Include appropriate controls (isotype-matched non-specific antibodies)

  • Determine optimal antibody concentration through dose-response experiments

  • Consider the timing of antibody addition relative to ligand stimulation

  • Verify inhibition through phosphorylation assays

• Therapeutic potential: Although there are clinically approved antibodies for ErbB1 and ErbB2, there are currently no available therapeutic monoclonal antibodies for ErbB4 . Research using neutralizing antibodies may help determine whether ERBB4 is a useful target for antibody-based cancer therapy.

What are the key storage and handling recommendations for ERBB4 antibodies?

Proper storage and handling are essential for maintaining antibody performance and extending shelf life:

• Storage temperature: Most ERBB4 antibodies should be stored at -20°C and are typically stable for one year after shipment under these conditions .

• Buffer composition: ERBB4 antibodies are often supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . This buffer composition helps maintain antibody stability.

• Aliquoting: For some antibody preparations, aliquoting may be unnecessary for -20°C storage, but for frequent use, creating small aliquots is recommended to avoid freeze-thaw cycles .

• BSA content: Some smaller volume preparations (e.g., 20μl sizes) may contain 0.1% BSA as a stabilizer .

• Working dilutions: Freshly prepared working dilutions should be used within the same day. Avoid storing diluted antibody solutions for extended periods.

• Avoiding contamination: Use sterile technique when handling antibody solutions to prevent microbial contamination that could degrade the antibody.

How do I troubleshoot common issues with ERBB4 antibody applications?

Researchers may encounter various challenges when working with ERBB4 antibodies. Here are solutions to common problems:

Western Blotting Issues:

• No signal or weak signal:

  • Increase antibody concentration (within recommended range)

  • Extend primary antibody incubation time

  • Ensure adequate protein loading (ERBB4 may be expressed at low levels in some samples)

  • Verify transfer efficiency for high molecular weight proteins

  • Check sample preparation (use phosphatase inhibitors for phospho-specific detection)

• Multiple bands:

  • Verify specificity using knockout controls

  • Consider the presence of different isoforms or cleavage products

  • Optimize blocking conditions to reduce non-specific binding

  • Ensure fresh samples (degradation may produce additional bands)

Immunohistochemistry Issues:

• High background:

  • Increase blocking time and concentration

  • Reduce primary antibody concentration

  • Optimize washing steps (increase number and duration)

  • Use lower secondary antibody concentration

• No staining or weak staining:

  • Try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Increase antibody concentration

  • Extend incubation times

  • Use an amplification system

  • Verify expression in the specific tissue type being examined

Immunoprecipitation Issues:

• Poor precipitation efficiency:

  • Increase antibody amount

  • Extend incubation time

  • Verify antibody compatibility with IP applications

  • Optimize lysis conditions to preserve native protein structure

• Contaminating bands in IP-Western:

  • Use TrueBlot or similar secondary antibodies that don't recognize denatured IgG

  • Consider using antibodies from different species for IP and Western detection