SH3TC2 Antibody, Biotin conjugated

What is SH3TC2 and why is it important in neurological research?

SH3TC2 (SH3 domain and tetratricopeptide repeat-containing protein 2, also known as KIAA1985) is a protein specifically expressed in Schwann cells that plays a critical role in peripheral nerve myelination. This protein has gained significant research interest because mutations in the SH3TC2 gene cause Charcot-Marie-Tooth disease type 4C (CMT4C), an autosomal recessive form of demyelinating neuropathy characterized by progressive scoliosis, delayed walking, muscular atrophy, and reduced nerve conduction velocity . SH3TC2 localizes to the plasma membrane and perinuclear endocytic recycling compartment, suggesting its role in membrane trafficking processes crucial for myelin maintenance .

What structural domains characterize the SH3TC2 protein?

SH3TC2 is a 144 kDa protein containing two N-terminal SH3 domains and at least six C-terminal tetratricopeptide repeat (TPR) motifs . Both domain types mediate protein-protein interactions. Additionally, SH3TC2 has a putative myristoylation site at the N-terminus (glycine at position 2) that is responsible for its plasma membrane localization, as demonstrated through in vitro myristoylation assays . Replacing this glycine with alanine (G2A) leads to loss of myristoylation capacity and altered cellular localization .

What are the typical applications for a biotin-conjugated SH3TC2 antibody?

Biotin-conjugated SH3TC2 antibodies are primarily used in ELISA applications as indicated in product specifications , but the biotin conjugation makes them versatile for multiple detection systems. Biotinylated antibodies can be detected using various streptavidin-conjugated reporter molecules (HRP, AP, fluorophores), offering flexibility in experimental design . They can potentially be used in Western blotting, immunohistochemistry, and immunofluorescence studies of Schwann cells and peripheral nerve samples, though specific validation for these applications may be necessary .

How should I optimize immunostaining protocols for SH3TC2 detection in peripheral nerve samples?

For optimal SH3TC2 detection in peripheral nerve samples:

• Tissue preparation: Ensure proper fixation (4% paraformaldehyde is commonly used) while maintaining epitope accessibility. For frozen sections, avoid repeated freeze-thaw cycles as indicated in storage recommendations for the antibody .

• Blocking: Implement thorough blocking with BSA and normal serum to minimize non-specific binding, particularly important given SH3TC2's specific expression in Schwann cells within a complex nerve environment .

• Detection system: Utilize the biotin-streptavidin system with appropriate amplification. Since SH3TC2 exhibits subcellular localization to both plasma membrane and endocytic compartments , a detection system with sufficient sensitivity to distinguish these compartments is essential.

• Controls: Include SH3TC2-knockout tissue (if available) as a negative control. Studies have generated knockout mice where the first exon of the Sh3tc2 gene is replaced with an enhanced GFP cassette , which can serve as excellent controls.

• Co-localization studies: Consider dual labeling with markers for the endocytic recycling compartment (e.g., Rab11) or Schwann cell-specific markers to confirm specificity and subcellular localization .

What are the critical considerations when using this antibody for studying SH3TC2's role in myelination disorders?

When investigating myelination disorders using biotin-conjugated SH3TC2 antibody:

• Developmental timing: SH3TC2 is expressed late during myelination and is downregulated following denervation . Therefore, the developmental stage of samples is crucial for accurate interpretation.

• Model selection: Consider models that recapitulate the disease phenotype. Sh3tc2-deficient mice develop progressive peripheral neuropathy with decreased motor and sensory nerve conduction velocity and hypomyelination .

• Cell type specificity: Since SH3TC2 is exclusively expressed in Schwann cells , ensure your experimental design accounts for this specificity when analyzing mixed cell populations.

• Protein interactions: SH3TC2 associates with integrin-α6 and interacts with Rab11 . Consider examining these interactions when studying the functional consequences of SH3TC2 mutations.

• Data interpretation: Transcriptomic studies have shown that SH3TC2 depletion affects genes involved in cell signaling, adhesion processes, and cholesterol biosynthesis . Correlate your findings with these known downstream effects.

How can I effectively use this biotin-conjugated antibody in co-immunoprecipitation experiments to identify novel SH3TC2 interaction partners?

For co-immunoprecipitation studies with biotin-conjugated SH3TC2 antibody:

• Optimization strategy:

  • Pre-clear lysates with unconjugated streptavidin beads to reduce non-specific binding

  • Perform antibody titration experiments to determine optimal antibody concentration

  • Consider chemical crosslinking to stabilize transient interactions

  • Include appropriate negative controls (e.g., non-specific biotin-conjugated IgG)

• Buffer considerations: Since SH3TC2 is membrane-associated via myristoylation , use lysis buffers containing mild detergents (0.5-1% NP-40 or Triton X-100) that preserve protein-protein interactions while effectively solubilizing membrane proteins.

• Elution approach: For biotin-conjugated antibodies, consider competitive elution with biotin or direct boiling in sample buffer. Alternatively, use TEV protease cleavage sites if incorporated into your experimental design.

• Validation of interactions: Confirm novel interactions through reciprocal co-immunoprecipitation and functional assays. Focus on proteins involved in endocytic recycling (particularly Rab11-related proteins) and integrin signaling pathways based on known SH3TC2 functions .

• Mass spectrometry analysis: Consider using quantitative proteomics approaches like SILAC or TMT labeling to identify and quantify SH3TC2 interactors with higher confidence.

What strategies can maximize signal detection sensitivity when using biotin-conjugated SH3TC2 antibody in microscopy applications?

To maximize signal detection with biotin-conjugated SH3TC2 antibody in microscopy:

• Signal amplification options:

  Amplification System	Advantages	Considerations
  Streptavidin-HRP with tyramide signal amplification	10-50× signal enhancement	Potential background increase
  Fluorescent streptavidin conjugates	Direct visualization	Lower amplification
  Streptavidin-biotin complexes	Increased binding sites	Risk of aggregate formation
  Quantum dot-streptavidin	Photostability, brightness	Cost, optimization required

• Confocal settings optimization: Since SH3TC2 localizes to specific subcellular compartments (plasma membrane and endocytic recycling compartment) , optimize pinhole size, detector gain, and laser power to enhance signal-to-noise ratio for these structures.

• Sample preparation: For optimal preservation of both membrane and endosomal structures, consider mild fixation protocols and permeabilization optimization (varying detergent types and concentrations).

• Multi-color imaging strategies: Implement co-localization studies with established markers for the endocytic recycling compartment (Rab11), plasma membrane, or Schwann cell markers to validate SH3TC2 localization patterns.

• Super-resolution approaches: Consider techniques like STED or STORM microscopy to resolve the precise localization of SH3TC2 within Schwann cell subcellular compartments, particularly at nodes of Ranvier where adhesion molecules play crucial roles .

How should I interpret discrepancies between SH3TC2 protein levels detected by this antibody and mRNA expression data?

When encountering discrepancies between SH3TC2 protein and mRNA levels:

• Regulatory mechanisms: Consider post-transcriptional regulation. SH3TC2 is regulated by transcription factors CREB and SOX10 , but protein levels may be further regulated by microRNAs, RNA-binding proteins, or protein stability mechanisms not reflected in mRNA measurements.

• Developmental timing: SH3TC2 shows dynamic expression, appearing late in myelination and downregulating following denervation . Ensure sample timing is aligned between protein and mRNA analyses.

• Experimental validation approach:

  • Perform time-course experiments to track both mRNA and protein expression

  • Use multiple antibodies targeting different epitopes to confirm protein expression patterns

  • Implement genetic models with tagged SH3TC2 to verify expression patterns independent of antibody detection

• Technical considerations: Biotin-conjugated antibodies may have different detection sensitivities compared to methods used for mRNA quantification. Normalize data appropriately and consider absolute quantification methods.

• Cellular heterogeneity: SH3TC2 is exclusively expressed in Schwann cells . Discrepancies might arise if comparing protein levels in purified Schwann cells versus mRNA from whole nerve tissue with varying cellular composition.

What controls are essential for validating specific SH3TC2 detection in complex neural tissues?

Essential controls for validating SH3TC2 detection include:

• Genetic negative controls: Tissue from Sh3tc2 knockout mice shows complete absence of the protein and develops peripheral neuropathy characteristic of CMT4C disease .

• Peptide competition assays: Pre-incubate the antibody with excess immunizing peptide (amino acids 188-427) to confirm signal specificity .

• Cellular specificity controls: Since SH3TC2 is exclusively expressed in Schwann cells , non-myelinating tissues should be negative. Denervated peripheral nerves should show downregulation .

• Alternative antibody validation: Compare results with other antibodies targeting different SH3TC2 epitopes, such as antibodies against amino acids 851-950 region mentioned in the search results .

• Signal validation in multiple assays: Confirm findings across complementary techniques (e.g., western blot, immunofluorescence, and immunohistochemistry) to ensure consistent detection of SH3TC2.

How can this antibody be used to study the role of SH3TC2 in Rab11-dependent trafficking pathways in Schwann cells?

To investigate SH3TC2's role in Rab11-dependent trafficking in Schwann cells:

• Live-cell imaging approach: Use the biotin-conjugated SH3TC2 antibody with cell-permeable streptavidin conjugates in live Schwann cell cultures to track dynamic trafficking events, particularly focusing on:

  • Co-localization with Rab11-positive vesicles

  • Trafficking of integrin-α6, a known SH3TC2-associated protein

  • Recycling dynamics of transferrin receptors, which are affected by SH3TC2

• Pulse-chase experimental design: Implement pulse-chase experiments with biotin-conjugated transferrin to assess endocytic recycling rates in normal versus SH3TC2-depleted Schwann cells, using the antibody to correlate recycling defects with SH3TC2 localization.

• Proximity labeling strategy: Combine with proximity labeling approaches (BioID or APEX) to identify proteins in close proximity to SH3TC2 within the endocytic recycling compartment.

• Super-resolution microscopy application: Apply techniques like STORM or STED to resolve the precise spatial relationship between SH3TC2, Rab11, and cargo proteins within the complex architecture of Schwann cell endosomes.

• Correlative light-electron microscopy (CLEM): Use the biotin-conjugated antibody with streptavidin-gold to precisely locate SH3TC2 within the ultrastructural context of the endocytic recycling compartment in Schwann cells.

What are the methodological approaches for investigating SH3TC2 expression changes during peripheral nerve injury and regeneration?

To study SH3TC2 dynamics during nerve injury and regeneration:

• Time-course experimental design:

  Time Point	Analysis Focus	Expected SH3TC2 Pattern
  Baseline	Normal expression	Membrane and endosomal localization
  Acute injury (1-3 days)	Downregulation	Rapid decrease following denervation
  Early regeneration (7-14 days)	Re-expression patterns	Potential correlation with remyelination
  Late regeneration (21-28 days)	Mature expression	Return to baseline if remyelination successful

• Spatial analysis approach: Implement whole-mount imaging or serial sectioning to examine SH3TC2 expression changes along the entire nerve, focusing on injury site, proximal and distal segments.

• Correlation with functional recovery: Combine immunodetection with electrophysiological measurements (nerve conduction velocity) and behavioral assessments to correlate SH3TC2 re-expression with functional recovery.

• Cellular context analysis: Perform co-labeling with markers for different stages of Schwann cell differentiation (c-Jun for repair Schwann cells, Krox20/Egr2 for myelinating Schwann cells) to correlate SH3TC2 expression with Schwann cell state transitions.

• Intervention studies: Use the antibody to track SH3TC2 expression following experimental manipulations that enhance or impair remyelination, thus establishing causative relationships between SH3TC2 dynamics and regenerative outcomes.

How can biotin-conjugated SH3TC2 antibodies facilitate the investigation of therapeutic interventions for CMT4C disease models?

For therapeutic intervention studies in CMT4C models:

• Biomarker validation strategy: Establish SH3TC2 detection protocols as pharmacodynamic biomarkers to assess therapy effects:

  • Quantify changes in SH3TC2 expression, localization, or interactor binding

  • Correlate these changes with functional improvements and myelin integrity

  • Use in conjunction with transcriptomic analysis of genes affected by SH3TC2 depletion

• High-content screening application: Implement the antibody in automated high-content screening platforms to evaluate compounds that might:

  • Enhance residual SH3TC2 function in cells expressing mutant forms

  • Upregulate compensatory pathways in SH3TC2-deficient cells

  • Promote endocytic recycling in Schwann cells independent of SH3TC2

• Gene therapy monitoring: Use the antibody to detect and quantify exogenously expressed SH3TC2 following gene therapy interventions, ensuring proper localization and function.

• Pharmacological validation: Assess whether compounds targeting the endocytic recycling pathway or integrin signaling can compensate for SH3TC2 deficiency, using the antibody to monitor effects on downstream pathways.

• Therapeutic window identification: Since SH3TC2 is expressed late in myelination , use temporal expression analysis to define the optimal therapeutic window for intervention in developmental or regenerative contexts.

SH3TC2 Antibody, Biotin Conjugated: Research Applications and Methodological Considerations

What are the key characteristics of commercially available biotin-conjugated SH3TC2 antibodies?

Commercially available biotin-conjugated SH3TC2 antibodies typically target specific amino acid sequences within the human SH3TC2 protein. For instance, several products target the region spanning amino acids 188-427 . These antibodies are generally:

• Raised in rabbit hosts

• Polyclonal in nature

• Purified using Protein G affinity purification (>95% purity)

• Formulated in buffers containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol, 0.01M PBS, pH 7.4)

• Validated primarily for ELISA applications

• Stored at -20°C or -80°C, with recommendations against repeated freeze-thaw cycles

The biotin conjugation provides significant advantages for detection flexibility, as these antibodies can be used with various streptavidin-conjugated detection systems (HRP, AP, fluorophores) depending on the experimental requirements .

What technical considerations are important when using biotin-conjugated antibodies for SH3TC2 detection?

When implementing biotin-conjugated SH3TC2 antibodies in research protocols, several technical considerations are essential:

• Endogenous biotin interference: Tissues and cells contain endogenous biotin that may interfere with detection. Consider using avidin/biotin blocking kits before applying biotinylated primary antibodies, particularly in biotin-rich tissues.

• Signal amplification options: The biotin-streptavidin system offers multiple amplification strategies:

  Amplification Method	Signal Enhancement	Application Suitability
  Direct streptavidin-reporter conjugates	1-4×	Western blotting, simple IHC
  ABC (Avidin-Biotin Complex)	4-8×	IHC, IF on tissue sections
  Tyramide signal amplification	10-50×	Low abundance targets, fluorescence

• Storage and handling: Avoid repeated freeze-thaw cycles as indicated in product specifications . Aliquot the antibody upon receipt to maintain conjugate stability.

• Validation controls: Include appropriate negative controls (biotin-conjugated non-specific IgG) and positive controls (tissues known to express SH3TC2, such as peripheral nerves). SH3TC2 knockout mouse tissues provide excellent negative controls .

• Background reduction: When high background is observed, optimize blocking (consider casein-based blockers rather than BSA to reduce biotin-binding protein interference) and include thorough washing steps with detergent-containing buffers.

How can biotin-conjugated SH3TC2 antibodies contribute to studying CMT4C pathophysiology?

Biotin-conjugated SH3TC2 antibodies serve as valuable tools for investigating CMT4C pathophysiology through multiple experimental approaches:

• Protein localization studies: SH3TC2 normally localizes to the plasma membrane through N-terminal myristoylation and to the endocytic recycling compartment . Pathogenic mutations disrupt this localization. Biotinylated antibodies can be used with high-resolution microscopy to characterize these mislocalization patterns in patient-derived cells or disease models.

• Functional interaction mapping: SH3TC2 interacts with Rab11 and affects the endocytic recycling of transferrin receptors . It also associates with integrin-α6, suggesting a role in Rab11-dependent endocytic trafficking of this laminin receptor in myelinated Schwann cells . Biotin-conjugated antibodies can be employed in proximity ligation assays to quantitatively assess these protein-protein interactions in normal versus disease conditions.

• Temporal expression analysis: Since SH3TC2 is expressed late during myelination , biotin-conjugated antibodies can help establish the precise developmental timing of SH3TC2 expression in relation to other myelin proteins and identify the optimal therapeutic window for intervention.

• Correlation with disease progression: In CMT4C models, these antibodies can track SH3TC2 expression and localization changes that correlate with disease progression, potentially identifying early biomarkers of pathology before clinical manifestations appear.

• Therapeutic response monitoring: Following experimental therapies aimed at restoring proper myelination in CMT4C models, these antibodies can assess whether SH3TC2 expression and localization patterns normalize in response to treatment.

What approaches can utilize biotin-conjugated SH3TC2 antibodies to explore membrane trafficking dynamics in Schwann cells?

Studying membrane trafficking dynamics in Schwann cells using biotin-conjugated SH3TC2 antibodies can be accomplished through several sophisticated approaches:

• Pulse-chase experiments: Using surface biotinylation followed by SH3TC2 immunoprecipitation to track the internalization and recycling rates of cell surface proteins in Schwann cells, particularly focusing on integrin-α6, which associates with SH3TC2 .

• Live-cell imaging: Employing cell-permeable fluorescent streptavidin conjugates with biotin-conjugated SH3TC2 antibodies to visualize trafficking events in real-time in cultured Schwann cells.

• Correlative light-electron microscopy (CLEM): Combining fluorescence microscopy using biotin-conjugated SH3TC2 antibodies with electron microscopy to precisely locate SH3TC2 within the ultrastructural context of the Schwann cell membrane and endosomal system.

• Super-resolution microscopy: Implementing techniques like STORM or STED with strategically labeled streptavidin to achieve nanoscale resolution of SH3TC2 localization within the complex architecture of the myelin sheath and nodes of Ranvier.

• Quantitative endosomal fractionation: Using biotin-conjugated SH3TC2 antibodies to immunoisolate specific endosomal subpopulations for proteomic analysis, helping to define the exact composition of SH3TC2-positive endosomes in Schwann cells.

What are common challenges in detecting SH3TC2 and how can they be addressed?

Researchers often encounter several challenges when detecting SH3TC2 using biotin-conjugated antibodies:

• Cell type specificity: Since SH3TC2 is exclusively expressed in Schwann cells , detection in mixed neural populations can be challenging. This can be addressed by:

  • Co-staining with Schwann cell-specific markers

  • Enriching for Schwann cells in mixed cultures

  • Using purified primary Schwann cell cultures or appropriate cell lines

• Low protein abundance: SH3TC2 may be expressed at low levels, particularly during specific developmental windows. Enhancement strategies include:

  • Using tyramide signal amplification with biotin-streptavidin systems

  • Employing photomultiplier amplification in confocal microscopy

  • Optimizing protein extraction protocols specifically for membrane proteins

• Background from endogenous biotin: Particularly problematic in biotin-rich tissues like brain and kidney. Solutions include:

  • Implementing avidin-biotin blocking steps before antibody application

  • Using alternative detection methods for verification

  • Including appropriate negative control samples (SH3TC2 knockout tissue)

• Epitope masking: Fixation may mask the epitope recognized by the antibody. Consider:

  • Testing multiple fixation protocols (paraformaldehyde, methanol, acetone)

  • Implementing antigen retrieval methods (heat-induced, enzymatic)

  • Using fresh-frozen sections instead of paraffin-embedded tissue

• Specificity verification: Confirm antibody specificity using:

  • Western blotting to verify the detection of a protein of the expected size (144 kDa)

  • Peptide competition assays using the immunizing peptide (amino acids 188-427)

  • Parallel testing with antibodies targeting different SH3TC2 epitopes

How should results from SH3TC2 antibody studies be interpreted in the context of functional data?

Proper interpretation of SH3TC2 antibody detection results requires integration with functional data:

• Correlation with myelination status: SH3TC2 expression correlates with myelination , so antibody detection should be interpreted alongside measurements of myelin thickness, g-ratio analysis, and expression of other myelin proteins.

• Relationship to Rab11 function: Since SH3TC2 is a Rab11 effector , changes in SH3TC2 detection should be considered in relation to Rab11 localization and recycling endosome function. Decreased colocalization may indicate disruption of this interaction.

• Integration with transcriptomic data: SH3TC2 depletion affects the expression of genes involved in cell signaling, adhesion processes, and cholesterol biosynthesis . Antibody detection findings should be interpreted alongside these transcriptomic changes.

• Developmental context: Given that SH3TC2 is expressed late in myelination , detection patterns must be interpreted in the appropriate developmental context, with particular attention to the timing of myelination in the model system being studied.

• Relationship to disease phenotypes: In CMT4C models, correlate SH3TC2 antibody detection patterns with physiological and structural measurements, including nerve conduction velocity, which is reduced in SH3TC2-deficient mice .

How can biotin-conjugated SH3TC2 antibodies contribute to therapeutic development for CMT4C?

Biotin-conjugated SH3TC2 antibodies can advance therapeutic development for CMT4C through several innovative approaches:

• Pharmacodynamic biomarker development: These antibodies can help establish quantifiable markers of drug effect in preclinical models, such as:

  • Restoration of proper SH3TC2 localization

  • Re-establishment of SH3TC2-Rab11 interactions

  • Normalization of endocytic recycling of key cargo like integrin-α6

• High-throughput screening platforms: Implement automated image-based screens using biotin-conjugated SH3TC2 antibodies to identify compounds that:

  • Correct mislocalization of mutant SH3TC2

  • Enhance residual function of mutant proteins

  • Upregulate compensatory pathways

• Gene therapy monitoring: In gene replacement approaches, these antibodies can verify:

  • Successful expression of exogenous SH3TC2

  • Proper localization to target compartments

  • Restoration of functional interactions with partner proteins

• Temporal therapeutic window definition: Since SH3TC2 is expressed late in myelination , these antibodies can help define the optimal timing for therapeutic intervention in developmental models or during regeneration attempts.

• Personalized medicine approaches: For patients with different SH3TC2 mutations, these antibodies could help categorize the precise molecular defects (e.g., expression level, localization, interaction disruption), potentially guiding mutation-specific therapeutic strategies.

What novel methodological combinations can enhance SH3TC2 research using biotin-conjugated antibodies?

Innovative methodological combinations can significantly advance SH3TC2 research:

• Expansion microscopy with biotin-streptavidin detection: Combining physical expansion of specimens with the specificity of biotin-conjugated SH3TC2 antibodies could reveal previously unappreciated details of SH3TC2 distribution within the intricate architecture of the myelin sheath.

• Single-molecule tracking in live Schwann cells: Using quantum dot-conjugated streptavidin with biotin-conjugated anti-SH3TC2 to track individual SH3TC2 molecules in living cells, providing insights into dynamic membrane trafficking events.

• Intravital imaging with biotin-conjugated antibodies: Employing biotin-conjugated SH3TC2 antibodies with in vivo compatible streptavidin-fluorophore conjugates for longitudinal imaging of SH3TC2 dynamics during development or in disease models.

• Multi-omics integration: Combining antibody-based imaging data with proteomics and transcriptomics through spatial transcriptomics or DISCo tissue clearing techniques to correlate SH3TC2 localization with gene expression patterns across peripheral nerve tissue.

• Optogenetic manipulation with antibody detection: Pairing optogenetic control of endosomal trafficking with SH3TC2 antibody detection to directly test hypotheses about SH3TC2 function in real-time while monitoring localization changes.