SH3TC2 Antibody, FITC conjugated

Basic Research Questions

• What is SH3TC2 and why is it significant for biomedical research?

  SH3TC2 is a protein-encoding gene that contains two N-terminal Src homology 3 domains and 10 tetratricopeptide repeat motifs . It plays a critical role as a Rab11 effector, targeting to the intracellular recycling endosome and regulating the recycling of internalized membrane and receptors back to the plasma membrane . SH3TC2 has dual significance in research: it is implicated in neurological disorders (particularly Charcot-Marie-Tooth disease type 4C) and has recently been identified as a potential oncogene in various cancers . The protein's involvement in multiple disease pathways makes it a valuable target for both basic research and therapeutic development.

• What are the optimal specimen preparation protocols for SH3TC2 antibody immunofluorescence?

  For optimal results with SH3TC2 antibody immunofluorescence, specimens should undergo careful fixation with 4% paraformaldehyde to preserve protein structure while maintaining epitope accessibility. Cell permeabilization should be performed using 0.1-0.2% Triton X-100 for intracellular targets. A comprehensive blocking step (5% normal serum with 1% BSA) is essential to minimize non-specific binding. When working with tissue sections, additional antigen retrieval may be necessary, typically using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For cultured cells, researchers should optimize cell density to ensure proper visualization of the recycling endosome structures where SH3TC2 predominantly localizes . Always include appropriate positive controls (such as colorectal cancer cell lines HCT116, SW480, or LOVO known to express SH3TC2) and negative controls (cells with SH3TC2 knockdown) .

• How does FITC conjugation affect SH3TC2 antibody performance?

  FITC (Fluorescein isothiocyanate) conjugation provides direct fluorescent labeling, eliminating the need for secondary antibody detection steps . While this streamlines workflow, FITC conjugation can influence antibody performance in several ways. The FITC fluorophore has excitation/emission maxima around 495/519 nm, producing green fluorescence that may overlap with cellular autofluorescence, particularly in certain tissue types . FITC is more susceptible to photobleaching compared to newer-generation fluorophores, requiring careful handling and imaging protocols. The conjugation process may occasionally affect antibody binding affinity or specificity, necessitating validation against unconjugated versions. Researchers should store FITC-conjugated antibodies at -20°C to -70°C under sterile conditions, protected from light, and avoid repeated freeze-thaw cycles to maintain optimal performance .

• What controls are essential when working with SH3TC2 antibody, FITC conjugated?

  A comprehensive control strategy for SH3TC2 antibody (FITC conjugated) experiments should include: (1) Isotype control - a FITC-conjugated antibody of the same isotype with no specific target, to assess non-specific binding; (2) Expression controls - negative controls using cells with confirmed SH3TC2 knockdown (via siRNA as described in colorectal cancer research) and positive controls using cell lines with confirmed SH3TC2 expression (HCT116, SW480, LOVO) ; (3) Peptide absorption control - pre-incubation of the antibody with immunizing peptide to confirm specificity; (4) Unstained sample control - to assess native autofluorescence; (5) Single-color controls - when performing multicolor experiments to establish compensation settings; and (6) Wild-type vs. mutant SH3TC2 expression systems - particularly when studying CMT4C-associated mutations, which affect protein localization . Quantitative analysis should be performed using standardized exposure settings across all samples.

• How can SH3TC2 antibody be validated for specificity?

  Rigorous validation of SH3TC2 antibody specificity should employ multiple complementary approaches: (1) Genetic validation - testing the antibody in cell lines with SH3TC2 knockdown (using siRNA or shRNA methods as described in colorectal cancer studies) or CRISPR-edited cell lines with SH3TC2 mutations ; (2) Western blot analysis - confirming a single band of appropriate molecular weight (approximately 144 kDa for full-length SH3TC2) in positive control samples and absence/reduction in negative controls ; (3) Peptide competition assay - pre-incubating the antibody with immunizing peptide should eliminate specific staining; (4) Cross-reactivity testing - evaluating staining patterns across multiple species if cross-reactivity is claimed; (5) Correlation with orthogonal methods - comparing antibody results with mRNA expression data or alternative antibodies targeting different epitopes; and (6) Expected localization pattern - confirming the antibody detects SH3TC2 primarily at recycling endosomes in wild-type cells and altered localization in cells expressing disease-associated mutations .

Experimental Design Considerations

• How should researchers design time-course experiments to study SH3TC2 dynamics?

  Effective time-course experiments for SH3TC2 dynamics should: (1) Establish appropriate temporal sampling - design time points based on the specific biological process being studied (minutes for acute signaling, hours for expression changes, days for phenotypic outcomes); (2) Implement synchronized starting conditions - use serum starvation/stimulation protocols or cell cycle synchronization methods to reduce variability; (3) Utilize pulse-chase approaches - for trafficking studies, combine with transferrin receptor dynamics experiments as SH3TC2 has been shown to influence these processes ; (4) Incorporate pathway perturbations - include MAPK pathway inhibitors/activators at defined time points since SH3TC2 functions through MAPK signaling in cancer contexts ; (5) Design parallel readout systems - simultaneously assess localization (immunofluorescence), expression (Western blot), and functional outcomes (proliferation, migration assays); (6) Include appropriate controls at each time point - particularly important when studying disease-associated mutations; (7) Implement quantitative image analysis - use consistent parameters for quantification across all time points; and (8) Correlate with clinical progression timescales when studying disease models - particularly important for both cancer progression and CMT4C pathology studies.

• What methodological approaches can distinguish between different functional domains of SH3TC2?

  To functionally characterize distinct SH3TC2 domains, researchers should implement: (1) Domain-specific antibody panel development - generate or obtain antibodies specifically targeting the N-terminal SH3 domains versus the TPR motifs; (2) Truncation construct analysis - systematically create and express SH3TC2 constructs containing specific domains to assess their individual contributions to localization and function; (3) Point mutation panels - introduce specific mutations in individual domains and assess effects on protein-protein interactions, particularly with Rab11 ; (4) Proximity labeling approaches - employ BioID or APEX2 fusion proteins to different SH3TC2 domains to identify domain-specific interaction partners; (5) Domain-focused co-immunoprecipitation - perform targeted pull-down experiments to identify proteins specifically interacting with each domain; (6) Comparative evolutionary analysis - assess conservation patterns across species to identify functionally critical regions within each domain; (7) Structural biology integration - incorporate available structural information to interpret functional data in a structural context; and (8) Disease mutation mapping - analyze the distribution of CMT4C-causing mutations across domains to identify functionally critical regions, particularly noting that mutations throughout SH3TC2 can cause similar clinical phenotypes .

  SH3TC2 Domain	Function	Disease Relevance	Detection Method
  SH3 domains (N-terminal)	Protein-protein interactions, potential role in signaling	Present in various CMT4C mutations	Domain-specific antibodies, truncation constructs
  Tetratricopeptide repeat motifs (10 repeats)	Mediates protein interactions, potential scaffolding role	Location of c.2860C>T (p.Arg954*) mutation	Domain-specific antibodies, deletion constructs
  Rab11 binding region	Critical for recycling endosome localization	Disrupted in CMT4C mutations	Co-IP, PLA, co-localization analysis
  MAPK pathway interaction domains	Cancer progression, invasion promotion	Relevant in colorectal cancer	Co-IP with MAPK8, 9, 13, 14

• How can researchers accurately quantify SH3TC2-mediated effects on endosomal trafficking?

  For precise quantification of SH3TC2's impact on endosomal trafficking, implement: (1) Transferrin receptor trafficking assays - measure internalization, recycling, and degradation rates in cells with wild-type versus mutant SH3TC2, as research has established that wild-type SH3TC2 influences transferrin receptor dynamics while mutant forms do not ; (2) Multi-parameter vesicle tracking - employ live-cell imaging with fluorescently labeled endosomal markers to measure vesicle size, movement velocity, directionality, and fusion/fission events; (3) Cargo-specific recycling assays - evaluate multiple receptor types beyond transferrin (e.g., integrins, growth factor receptors) to assess cargo specificity; (4) Rab11 activity measurements - implement FRET-based Rab11 activity sensors to correlate SH3TC2 effects with Rab11 activation state ; (5) High-content screening platforms - develop automated image analysis pipelines for large-scale quantification of trafficking parameters; (6) Compartment-specific marker analysis - systematically assess distribution of early endosome, recycling endosome, late endosome, and lysosome markers; (7) Super-resolution time-lapse microscopy - capture nanoscale dynamics of endosomal structures; and (8) Correlative light-electron microscopy - combine functional fluorescence data with ultrastructural analysis of affected compartments.