SH3TC2 Antibody, HRP conjugated

What is SH3TC2 protein and why is it significant for neurological research?

SH3TC2 (SH3 domain and tetratricopeptide repeat-containing protein 2) is a 144 kDa protein with two N-terminal SH3 domains and five C-terminal TPR motifs that facilitate protein-protein interactions . It is predominantly expressed in Schwann cells of the peripheral nervous system and plays an essential role in peripheral nerve myelination . The significance of SH3TC2 in neurological research stems from its association with Charcot-Marie-Tooth disease type 4C (CMT4C), a severe hereditary peripheral neuropathy . SH3TC2 functions as a Rab11 effector at the recycling endosome, regulating the recycling of internalized membrane and receptors back to the plasma membrane . Mutations in SH3TC2 disrupt this interaction, leading to intracellular mistargeting away from the recycling endosome and consequent impairment in myelin formation .

What are the key specifications of SH3TC2 Antibody, HRP conjugated?

The SH3TC2 Antibody, HRP conjugated (e.g., PACO55215) is a polyclonal antibody sourced from rabbit with an IgG isotype . It demonstrates reactivity against human samples and is specifically designed for ELISA applications . The antibody is raised against a recombinant human SH3TC2 protein fragment (amino acids 188-427) . The horseradish peroxidase (HRP) conjugation facilitates direct detection in immunoassays without requiring secondary antibodies . Typically available in 50μg quantities, the antibody is stored in a preservative solution containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 .

How does SH3TC2 function in cellular trafficking pathways?

SH3TC2 functions primarily at the recycling endosome, where it associates with the small GTPase Rab11 in a GTP-dependent manner . As a Rab11 effector, SH3TC2 regulates the recycling of internalized proteins and membrane components back to the cell surface . The protein's wild-type form targets to tubulovesicular structures concentrated near the nucleus but distributed throughout the cell, displaying a distinct localization pattern emanating from the perinuclear compartment and extending to the plasma membrane . SH3TC2 specifically influences transferrin receptor dynamics, with overexpression of wild-type SH3TC2 decreasing cell surface concentration of transferrin receptors . This mechanism appears especially important in Schwann cells, where SH3TC2 may regulate the recycling of specific membrane receptors crucial for myelination processes .

What are the optimal conditions for using SH3TC2 Antibody, HRP conjugated in ELISA?

When designing ELISA experiments with SH3TC2 Antibody, HRP conjugated, researchers should implement the following methodological approach:

• Coating phase: Use purified recombinant SH3TC2 protein or cell lysates containing SH3TC2 at 1-10 μg/ml in carbonate buffer (pH 9.6) for coating plates.

• Blocking: Block non-specific binding sites with 3-5% BSA or non-fat milk in PBS-T (PBS with 0.05% Tween-20) for 1-2 hours at room temperature.

• Primary antibody: Apply SH3TC2 Antibody, HRP conjugated at titered dilutions ranging from 1:500 to 1:5000 in blocking buffer. Incubate for 1-2 hours at room temperature or overnight at 4°C.

• Washing: Perform 4-5 wash cycles with PBS-T between each step to minimize background.

• Detection: Add TMB substrate and monitor color development. Stop the reaction with 2N H₂SO₄ and read absorbance at 450 nm.

• Controls: Include no-antigen, no-primary antibody, and positive control (known SH3TC2-positive samples) conditions to validate results .

For quantitative assays, prepare a standard curve using purified SH3TC2 protein at known concentrations (0.1-1000 ng/ml) to accurately determine SH3TC2 levels in experimental samples.

How can researchers validate SH3TC2 Antibody specificity for studying Rab11-SH3TC2 interactions?

Validating SH3TC2 Antibody specificity for studying Rab11-SH3TC2 interactions requires a multi-step approach:

• Western blot verification: Perform western blots on lysates from cells expressing wild-type SH3TC2 versus SH3TC2 with CMT4C-associated mutations. The antibody should detect the expected 144 kDa band in wild-type samples.

• Immunoprecipitation assays: Use the antibody to immunoprecipitate SH3TC2, then probe for co-precipitated Rab11 using anti-Rab11 antibodies. This should show enrichment of Rab11 in GTP-loaded conditions compared to GDP-loaded conditions .

• GST pull-down assays: As described in previous research, perform GST-Rab11 pull-down assays with cell lysates containing GFP-SH3TC2 WT and mutant constructs. The antibody should detect SH3TC2 in pulled-down fractions from wild-type but not mutant samples .

• Co-localization studies: Use the antibody in immunofluorescence studies alongside markers for Rab11 and recycling endosomes. Wild-type SH3TC2 should show strong co-localization with Rab11, while mutant forms should not .

• Competition assays: Pre-incubate the antibody with recombinant SH3TC2 protein before use in any detection method to confirm that signal loss occurs, confirming specificity.

Each validation step should include appropriate controls, including isotype controls and samples expressing mutant SH3TC2 known to disrupt Rab11 binding .

What experimental design is recommended for investigating SH3TC2's role in transferrin receptor trafficking?

To investigate SH3TC2's role in transferrin receptor trafficking, researchers should implement the following experimental design:

Study Design Table: SH3TC2 and Transferrin Receptor Trafficking

Methodological approach:

• FACS analysis: Transfect cells with GFP-tagged wild-type or mutant SH3TC2 constructs. After 24-48 hours, stain non-permeabilized cells with anti-transferrin receptor antibody. Use flow cytometry to quantify surface TfR levels specifically in GFP-positive cells .

• Transferrin uptake and recycling assays: Incubate transfected cells with fluorescently-labeled transferrin for 30 minutes at 37°C, then chase with unlabeled transferrin. Measure fluorescence at various time points to track internalization and recycling rates .

• Live-cell imaging: Perform time-lapse microscopy of cells co-expressing fluorescently tagged SH3TC2 and transferrin receptor to visualize trafficking dynamics in real-time.

• siRNA knockdown: Deploy RNA interference to deplete endogenous SH3TC2 and measure effects on transferrin receptor surface levels and trafficking. Include rescue experiments with wild-type and mutant constructs .

This comprehensive approach allows for quantitative assessment of how wild-type versus mutant SH3TC2 differentially influences transferrin receptor dynamics, providing insights into the protein's physiological function .

How can researchers use SH3TC2 Antibody to investigate pathogenic mechanisms in Charcot-Marie-Tooth disease type 4C?

Investigating pathogenic mechanisms in CMT4C using SH3TC2 Antibody, HRP conjugated requires a sophisticated experimental approach:

• Patient-derived materials: Utilize the antibody to compare SH3TC2 expression and localization in peripheral nerve biopsies or cultured Schwann cells from CMT4C patients versus healthy controls. This can reveal whether mutant proteins are expressed but mislocalized, or degraded in patient samples.

• Transgenic animal models: Apply immunohistochemistry with the antibody to analyze SH3TC2 distribution in transgenic mice expressing human wild-type or mutant SH3TC2. Focus on sciatic nerve sections and dorsal root ganglia to assess Schwann cell-specific localization .

• Co-immunoprecipitation studies: Use the antibody to pull down SH3TC2 from lysates of cells expressing wild-type or mutant constructs, then analyze binding partners by mass spectrometry to identify differentially interacting proteins beyond just Rab11.

• Pulse-chase experiments: Employ metabolic labeling combined with immunoprecipitation using the SH3TC2 antibody to track protein stability and turnover rates of wild-type versus mutant proteins.

• Proximity ligation assays: Utilize the SH3TC2 antibody in conjunction with Rab11 antibodies to visualize and quantify endogenous SH3TC2-Rab11 interactions in situ, comparing normal versus pathogenic contexts.

This multifaceted approach can elucidate how mutations disrupt SH3TC2's interaction with Rab11 and other potential binding partners, providing insight into the molecular basis of CMT4C pathogenesis .

What methodological approach should be used to investigate the N-terminal targeting domain (amino acids 1-50) of SH3TC2?

To investigate the N-terminal targeting domain (amino acids 1-50) of SH3TC2, researchers should implement the following methodological approach:

• Truncation and mutation analysis: Generate a series of constructs with progressive truncations and point mutations within the N-terminal 1-50 amino acid region. Express these in relevant cell types alongside full-length wild-type controls .

• Subcellular localization studies: Use confocal microscopy to assess localization patterns of each construct, paying particular attention to recycling endosome markers like Rab11. Previous research has shown that deletion of the first 50 amino acids (Δ1-50) maintains normal localization, suggesting complexity in targeting mechanisms .

• Protein-lipid interaction assays: Perform lipid overlay assays and liposome binding experiments with purified N-terminal fragments to test for potential direct membrane interactions, as myristoylation at G2 has been previously hypothesized but not confirmed as essential for function .

• Structure-function analysis: Employ circular dichroism and NMR to determine structural elements within the N-terminal domain that might contribute to protein folding or interaction surfaces.

• Interaction mapping: Use the SH3TC2 antibody in immunoprecipitation experiments with various N-terminal constructs to identify which regions are essential for Rab11 binding. Complement with yeast two-hybrid or mammalian two-hybrid assays.

How can researchers design experiments to analyze the GTP-dependence of the SH3TC2-Rab11 interaction?

To analyze the GTP-dependence of the SH3TC2-Rab11 interaction, researchers should design experiments following this methodological framework:

Experimental design for GTP-dependence analysis:

• GST pull-down assays with nucleotide loading:

  • Express and purify GST-tagged Rab11 protein

  • Pre-load GST-Rab11 with either GTPγS (non-hydrolyzable GTP analog), GDP, or no nucleotide

  • Incubate with cell lysates containing GFP-tagged wild-type SH3TC2

  • Analyze pulled-down fractions by western blotting using SH3TC2 antibody

  • Quantify relative binding efficiency across nucleotide conditions

• Co-immunoprecipitation with Rab11 mutants:

  • Transfect cells with myc-tagged Rab11 mutants (Q70L for constitutively active/GTP-bound; S25N for dominant negative/GDP-bound)

  • Co-express with wild-type SH3TC2

  • Immunoprecipitate using anti-myc antibody

  • Probe for co-precipitated SH3TC2 using the SH3TC2 antibody

  • Compare binding efficiency between different Rab11 mutants

• Fluorescence microscopy with Rab11 mutants:

  • Co-express GFP-SH3TC2 with myc-Rab11 Q70L or myc-Rab11 S25N

  • Analyze co-localization patterns using confocal microscopy

  • Quantify Pearson's correlation coefficients to measure degree of co-localization

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC):

  • Purify recombinant SH3TC2 and Rab11 proteins

  • Perform binding kinetics analysis with Rab11 pre-loaded with different nucleotides

  • Determine binding affinities (Kd values) for each nucleotide state

These complementary approaches will establish whether SH3TC2 preferentially binds to GTP-bound Rab11 (characteristic of effector proteins) and quantify the specificity of this interaction .

What strategies should researchers employ when facing inconsistent results in SH3TC2 localization studies?

When researchers encounter inconsistent results in SH3TC2 localization studies, they should implement the following systematic troubleshooting approach:

• Validate antibody specificity: Test the SH3TC2 antibody on western blots of lysates from cells with confirmed SH3TC2 expression versus knockout/knockdown cells. Ensure that a single band of the expected molecular weight (144 kDa) is detected .

• Optimize fixation methods: Different fixation protocols can significantly impact antibody accessibility and protein localization. Compare paraformaldehyde (4%, 10-15 minutes) versus methanol fixation (-20°C, 10 minutes) to determine optimal conditions for preserving SH3TC2's native localization at the recycling endosome.

• Use multiple markers for recycling endosomes: Co-stain with additional markers beyond Rab11, such as transferrin receptor and EHD1, to confirm genuine recycling endosome localization .

• Compare expression levels: Overexpression can lead to artifactual localization. Use systems with tunable expression (e.g., tetracycline-inducible) to test whether localization patterns change at different expression levels.

• Evaluate cell type-specific differences: Reports have shown potentially different localizations in different cell types. Systematically compare SH3TC2 localization in Schwann cells, HeLa cells, and other relevant cell types using identical protocols .

• Consider splice variants or post-translational modifications: Investigate whether alternative splicing or post-translational modifications could explain observed differences in localization patterns.

By implementing this structured approach, researchers can resolve discrepancies between different reports, such as the conflicting localizations described in the literature where some studies reported plasma membrane localization while others documented specific recycling endosome targeting .

How should researchers interpret SH3TC2 antibody signals in the context of mutant protein studies?

Interpreting SH3TC2 antibody signals in mutant protein studies requires careful consideration of several factors:

• Protein expression levels: When comparing wild-type versus mutant SH3TC2, always quantify total protein expression by western blot to ensure observed differences in localization or function are not simply due to expression disparities .

• Epitope accessibility: Mutations might alter protein conformation, potentially affecting antibody binding. Use multiple antibodies targeting different epitopes to confirm results, especially when studying CMT4C-associated mutations .

• Distinguishing cytosolic from membrane-associated signals: Wild-type SH3TC2 shows distinct tubulovesicular localization, while pathogenic mutants display diffuse cytosolic distribution. When quantifying images, use line-scan analysis across cellular regions to objectively measure signal distribution patterns .

• Analysis framework for interpreting localization data:

  Localization Pattern	Interpretation	Further Validation
  Tubulovesicular perinuclear	Functional recycling endosome targeting	Co-localization with Rab11
  Diffuse cytosolic	Loss of targeting ability	GST-Rab11 pull-down
  Plasma membrane	Potential over-expression artifact	Titrate expression levels
  Partial/intermediate	Hypomorphic mutation	Functional assays (TfR recycling)

• Correlation with functional data: Always correlate localization findings with functional assays, such as transferrin receptor dynamics or Rab11 binding studies, to establish biological significance of observed patterns .

This structured analytical approach allows researchers to confidently interpret whether specific mutations cause complete loss of function or partial defects in SH3TC2 targeting and activity .

What are the key considerations when analyzing contradictory data regarding SH3TC2 myristoylation at G2?

When analyzing contradictory data regarding SH3TC2 myristoylation at G2, researchers should consider the following methodological approach:

By systematically addressing these considerations, researchers can resolve the apparent contradiction between predicted myristoylation and experimental data showing that the N-terminal region is dispensable for core protein function .

How does studying SH3TC2-Rab11 interaction contribute to understanding the molecular pathogenesis of peripheral neuropathies?

The study of SH3TC2-Rab11 interactions provides critical insights into peripheral neuropathy pathogenesis through several interconnected mechanisms:

• Membrane trafficking in myelination: SH3TC2 functions as a Rab11 effector at the recycling endosome, regulating the recycling of internalized membrane components back to the cell surface . In Schwann cells, this process is crucial for myelin maintenance, as myelination requires continuous membrane remodeling and receptor recycling .

• Mechanistic link between mutations and disease: All CMT4C-associated mutations in SH3TC2 (both nonsense and missense) disrupt Rab11 binding and recycling endosome localization, providing a unifying mechanistic explanation for how diverse mutations throughout the protein cause the same clinical phenotype .

• Cell type-specific vulnerability: SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, explaining why mutations selectively affect peripheral nerves while sparing central nervous system myelin .

• Functional consequences in model systems: In vitro studies demonstrate that disruption of SH3TC2-Rab11 interaction or expression of dominant-negative Rab11 strongly impairs myelin formation, establishing a causal link between endosomal recycling and myelination .

• Therapeutic implications: Understanding that CMT4C pathogenesis involves disrupted endosomal recycling opens avenues for therapeutic intervention, such as enhancing alternative recycling pathways or stabilizing myelin without requiring the SH3TC2-Rab11 interaction.

This research establishes endosomal recycling as a crucial cellular mechanism for peripheral nerve myelination and maintenance, expanding our understanding of the cellular processes that can be dysregulated in peripheral neuropathies .

What are the key methodological approaches for investigating SH3TC2 function in primary Schwann cells versus cell lines?

Investigating SH3TC2 function requires distinct methodological approaches when working with primary Schwann cells versus cell lines:

Comparative Methodological Framework:

Key methodological considerations:

• Expression patterns: Endogenous SH3TC2 expression is readily detectable in primary Schwann cells but minimal in most cell lines. Antibody sensitivity requirements differ accordingly .

• Morphological analysis: In primary Schwann cells, analyze SH3TC2's distribution in relation to myelin structures using myelin markers (MBP, P0). In cell lines, focus on co-localization with endosomal markers .

• Functional readouts: For primary Schwann cells, assess myelination capacity (myelin segment formation) as the ultimate functional readout. For cell lines, transferrin receptor trafficking serves as a surrogate marker of function .

• Data integration: Combine observations from both systems—mechanistic insights from cell lines with physiological relevance from primary cells—to build comprehensive understanding of SH3TC2 function.

This dual approach leverages the complementary strengths of each system: cell lines for molecular mechanism studies and primary cells for disease-relevant functional analyses .

How might understanding the SH3TC2-Rab11 interaction pathway inform therapeutic strategies for Charcot-Marie-Tooth disease type 4C?

Understanding the SH3TC2-Rab11 interaction pathway provides several avenues for developing therapeutic strategies for CMT4C:

• Small molecule modulators: Develop compounds that could enhance residual SH3TC2-Rab11 interactions in patients with missense mutations, potentially stabilizing partially functional complexes.

• Alternative recycling pathway enhancement: Since direct restoration of SH3TC2 function may be challenging, upregulating compensatory endocytic recycling pathways (e.g., Rab4-dependent fast recycling) could bypass the need for SH3TC2-mediated recycling in Schwann cells .

• Targeted gene therapy approaches:

  • Gene replacement: Deliver functional SH3TC2 specifically to Schwann cells using viral vectors with cell-type specific promoters

  • Gene editing: Correct specific mutations in patient-derived cells, potentially applicable for autologous cell therapy

• Recycling endosome stabilization: Develop compounds that stabilize recycling endosome function in the absence of proper SH3TC2 function, focusing on the transferrin receptor pathway as a model system for evaluation .

• Downstream effector targeting: Identify and target critical cargo proteins whose recycling is particularly dependent on SH3TC2-Rab11 interaction in Schwann cells, potentially including:

  • Neuregulin receptors (ErbB2/3) which regulate myelination

  • Cell adhesion molecules required for Schwann cell-axon interactions

  • Lipid transporters needed for myelin membrane maintenance

This pathway-focused approach is particularly promising because SH3TC2 expression is restricted to Schwann cells in the peripheral nervous system, potentially allowing for targeted interventions with minimal off-target effects in other tissues .