SHC4 Antibody

What is SHC4 and what cellular functions does it serve?

SHC4 (also known as SHCD) is a member of the SHC family of adaptor proteins characterized by two phosphotyrosine-interaction modules: an amino-terminal phosphotyrosine binding (PTB) domain and a carboxy-terminal Src homology 2 domain . Functionally, SHC4 activates both Ras-dependent and Ras-independent migratory pathways in melanomas and contributes to the early phases of agrin-induced tyrosine phosphorylation of CHRNB1 . Unlike other SHC family proteins, SHC4 is primarily expressed in adult brain and skeletal muscle tissues, suggesting non-redundant functions compared to other family members .

What are the structural features and molecular weight of SHC4?

SHC4 has a calculated molecular weight of 69 kDa with 630 amino acids . The protein contains characteristic domains of the SHC family, including:

• An N-terminal phosphotyrosine binding (PTB) domain

• A central CH1 (collagen homology 1) region

• A C-terminal SH2 (Src homology 2) domain

The protein possesses unique phosphorylation sites, including a novel Grb2 binding site not found in other SHC family proteins . These structural features facilitate its role as a phosphotyrosine adaptor molecule in various receptor-mediated signaling pathways .

How should SHC4 antibodies be stored for optimal stability?

According to multiple antibody manufacturers, optimal storage conditions for SHC4 antibodies are:

For optimal performance, aliquot antibodies upon receipt and avoid repeated freezing and thawing, which can lead to antibody degradation and reduced performance in experimental applications .

Experimental Applications

SHC4 has been successfully detected in:

Tissue samples with positive detection:

• Human brain tissue

• Human skeletal muscle tissue

• Mouse brain tissue

• Rat brain tissue

• Human liver cancer tissue

• Human cervical cancer tissue

Cell lines with positive detection:

• SK-MEL-30 cells

• SH-SY5Y cells

• HepG2 cells

• A375 cells

• U2OS cells

For optimal results when studying SHC4, researchers should select these validated tissue types or cell lines as positive controls in their experiments .

How can I optimize immunohistochemistry protocols for SHC4 detection?

For optimal IHC detection of SHC4, consider these methodological approaches:

• Antigen retrieval methods:

  • Primary recommendation: Use TE buffer at pH 9.0

  • Alternative approach: Citrate buffer at pH 6.0

• Fixation and permeabilization:

  • PFA fixation followed by Triton X-100 permeabilization has been validated for cellular staining

• Antibody concentration:

  • Initial dilution range: 1:50-1:500 for IHC applications

  • For paraffin-embedded tissues: 1:90 dilution has been validated

• Visualization system:

  • Chromogenic detection systems are commonly used for tissue samples

  • For cellular samples, fluorescent secondary antibodies with appropriate filters should be used

Always include positive control tissues (brain or skeletal muscle) and negative controls (isotype control or secondary antibody only) to validate staining specificity .

What are the considerations for using SHC4 antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation (CoIP) experiments to study SHC4 interactions:

• Antibody selection:

  • Choose antibodies validated for IP applications

  • Consider the epitope location to avoid interference with protein-protein interaction sites

• Lysate preparation:

  • For membrane-associated complexes, use mild detergents (0.5-1% NP-40 or Triton X-100)

  • Include phosphatase inhibitors to preserve phosphorylation-dependent interactions

  • Maintain cold temperatures throughout to preserve protein complexes

• Interaction partners to consider:

  • MuSK (muscle-specific kinase) receptor tyrosine kinase, which associates with ShcD via its PTB domain

  • Components of Ras-dependent and Ras-independent signaling pathways

• Controls:

  • Use IgG isotype control to identify non-specific binding

  • Consider reverse CoIP to confirm interactions

  • Include input samples (5-10% of lysate) to confirm protein expression

These methodological considerations will help ensure reliable results when investigating SHC4's protein interaction network .

How should I design experiments to investigate SHC4's role in melanoma migration?

To study SHC4's role in melanoma migration, consider this experimental approach:

• Model selection:

  • Cell lines: A375 or SK-MEL-30 cells show reliable SHC4 expression

  • Patient-derived xenografts or tissue samples for clinical relevance

• Functional assessment methods:

  • Transwell migration assays to quantify cell migration

  • Wound healing assays for directional migration

  • Time-lapse microscopy for real-time migration tracking

• Mechanistic investigation:

  • Knockdown/knockout studies using siRNA or CRISPR/Cas9

  • Rescue experiments with wild-type vs. mutant SHC4

  • Phosphorylation analysis using phospho-specific antibodies

  • Downstream pathway analysis (Ras-dependent and Ras-independent)

• Controls and validation:

  • Verify knockdown/knockout efficiency by Western blot

  • Confirm antibody specificity using knockout controls

  • Include positive controls (known migration stimulators)

This comprehensive approach will enable detailed analysis of both SHC4-dependent migratory capacity and the underlying signaling mechanisms in melanoma cells .

What are the best approaches for studying SHC4 phosphorylation status?

Studying SHC4 phosphorylation requires specialized techniques:

• Antibody-based detection:

  • Western blotting with phospho-specific antibodies (if available)

  • Immunoprecipitation followed by phospho-tyrosine antibody detection

  • Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

• Stimulation conditions:

  • Serum starvation followed by growth factor stimulation

  • For muscle-specific studies, agrin stimulation (known to induce tyrosine phosphorylation downstream of MuSK)

  • Time course experiments to capture transient phosphorylation events

• Mass spectrometry approaches:

  • Enrichment of phosphopeptides using TiO₂ or IMAC

  • Targeted MS/MS for specific phosphorylation sites

  • Quantitative phosphoproteomics for comprehensive site mapping

• Data analysis:

  • Normalization to total SHC4 protein levels

  • Kinetic analysis of phosphorylation/dephosphorylation

  • Correlation with functional outcomes

These approaches will provide insights into how phosphorylation regulates SHC4's adaptor function in different signaling contexts .

How can I validate the specificity of my SHC4 antibody?

Thorough antibody validation is critical for reliable results:

• Western blot validation:

  • Verify the observed molecular weight matches the expected 69 kDa

  • Test multiple tissue types, including positive controls (brain, skeletal muscle) and negative controls

  • Include knockdown/knockout samples if available

• Cross-reactivity assessment:

  • Test antibody against other SHC family members (SHC1/2/3) to confirm specificity

  • Assess performance in tissues from different species based on documented reactivity

• Immunogen comparison:

  • Review the immunogen used to generate the antibody

  • Many SHC4 antibodies use amino acids 50-150 or 1-185 as immunogens

  • Compare with the region of interest in your study

• Confirmatory approaches:

  • Use multiple antibodies targeting different epitopes

  • Compare results between different detection methods (WB, IF, IHC)

  • Genetic validation through RNA interference or gene editing

Following these validation steps ensures confidence in experimental findings and helps troubleshoot unexpected results .

What are common pitfalls when using SHC4 antibodies, and how can they be avoided?

Researchers should be aware of these common challenges:

• Signal specificity issues:

  • Problem: Non-specific background or multiple bands

  • Solution: Optimize blocking conditions (5% BSA often superior to milk for phospho-proteins); increase antibody dilution; include knockout controls

• Inconsistent detection:

  • Problem: Variable results between experiments

  • Solution: Use recombinant antibodies for batch-to-batch consistency ; standardize lysate preparation; ensure constant protein loading

• Tissue-specific challenges:

  • Problem: Poor signal in tissues known to express SHC4

  • Solution: Optimize antigen retrieval methods for IHC (TE buffer pH 9.0 recommended) ; adjust fixation protocols; test multiple antibody clones

• Species cross-reactivity limitations:

  • Problem: Antibody may not work across all species despite homology

  • Solution: Confirm species reactivity in product data; test antibodies specifically validated for your species of interest

• Phosphorylation-dependent epitope masking:

  • Problem: Reduced detection of phosphorylated SHC4

  • Solution: Include phosphatase inhibitors in lysis buffers; compare results with and without phosphatase treatment

Addressing these common pitfalls proactively will improve experimental reliability and data interpretation .

How is SHC4 antibody being used to study neurological disorders?

SHC4's prominent expression in brain tissue makes it relevant for neurological research:

• Expression profiling:

  • SHC4 antibodies have been validated for detecting expression in human, mouse, and rat brain tissues

  • Useful for comparative studies across different neurological conditions

• Localization studies:

  • SHC4 localizes to cell junctions and synapses (specifically postsynaptic cell membranes)

  • Co-localization with synaptic markers can provide insights into neuronal function

• Neuromuscular junction research:

  • SHC4 co-localizes with MuSK at the neuromuscular junction

  • Contributes to agrin-induced tyrosine phosphorylation of CHRNB1 (acetylcholine receptor β subunit)

• Methodological approaches:

  • IHC on brain sections (1:50-1:500 dilution recommended)

  • Primary neuronal cultures for IF studies

  • Biochemical fractionation to isolate synaptic compartments

These applications enable investigation of SHC4's potential roles in synaptic plasticity, neurodegenerative disorders, and neuromuscular junction formation .

What is the current understanding of SHC4's role in cancer, and how are antibodies advancing this research?

SHC4 has emerging significance in cancer research:

• Expression pattern in cancer:

  • Detected in human liver cancer and cervical cancer tissues

  • Expression in melanoma cell lines (A375, SK-MEL-30)

• Functional role:

  • Activates both Ras-dependent and Ras-independent migratory pathways in melanomas

  • Potential contributor to cancer cell migration and invasion

• Research applications:

  • Expression profiling across cancer types using tissue microarrays

  • Correlation of expression levels with clinical outcomes

  • Mechanistic studies of SHC4-dependent signaling in cancer progression

• Methodological considerations:

  • For cancer tissue IHC: 1:90 dilution has been validated

  • Melanoma cell models: A375 and SK-MEL-30 cells show reliable expression

  • Functional studies should include migration and invasion assays

Further investigation using SHC4 antibodies may reveal potential prognostic significance or therapeutic targeting opportunities in various cancer types .

How can I combine SHC4 antibodies with other techniques for comprehensive signaling pathway analysis?

Integrating multiple techniques provides deeper insights into SHC4 biology:

• Multi-omics approaches:

  • Immunoprecipitation followed by mass spectrometry (IP-MS) to identify novel interaction partners

  • ChIP-seq following SHC4 pathway activation to identify transcriptional changes

  • Phosphoproteomics to map signaling cascade downstream of SHC4

• Advanced imaging methods:

  • Proximity ligation assay (PLA) to visualize SHC4 interactions in situ

  • FRET/FLIM to study dynamic protein interactions

  • Super-resolution microscopy for precise subcellular localization

• Functional genomics integration:

  • CRISPR screens targeting SHC4 pathway components

  • Single-cell analysis to capture heterogeneity in SHC4 expression and function

  • Correlation between genomic alterations and SHC4 pathway activation

• Quantitative techniques:

  • Cytometric bead arrays for sensitive SHC4 quantification (validated range: 0.781-100 ng/mL)

  • Multiplex Western blotting for simultaneous pathway component analysis

  • High-content imaging for multi-parameter phenotypic analysis

These integrated approaches will advance understanding of SHC4's role in complex signaling networks .

What are the emerging technologies for studying SHC4 that researchers should be aware of?

Cutting-edge approaches for SHC4 research include:

• Recombinant antibody technologies:

  • Matched antibody pairs for sensitive cytometric bead arrays

  • Recombinant antibodies for enhanced reproducibility and reduced batch variation

  • Site-specific conjugation for improved imaging applications

• Genetic engineering approaches:

  • CRISPR/Cas9 knock-in of tags for endogenous SHC4 tracking

  • Optogenetic control of SHC4 signaling pathways

  • Domain-specific mutagenesis to dissect functional regions

• Structural biology integration:

  • Cryo-EM studies of SHC4 in signaling complexes

  • Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

  • Computational modeling of SHC4 interactions based on structural data

• Translational research tools:

  • Patient-derived organoids for studying SHC4 in disease contexts

  • High-throughput drug screening targeting SHC4-dependent pathways

  • In vivo imaging of SHC4 activity using antibody-based probes

Researchers should consider these emerging technologies when designing comprehensive studies of SHC4 function in normal physiology and disease states .