toca-1 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Toca-1 is involved in a novel pathway that functions in parallel with a SUN-KASH nuclear envelope bridge to move nuclei in Caenorhabditis elegans. PMID: 23150597
Q&A
How to validate TOCA-1 antibody specificity in epithelial junction studies?
Methodological approach:
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Perform CRISPR-mediated TOCA-1 knockout in MDCK cells as negative controls. Compare immunofluorescence patterns between wild-type and knockout cells (loss of signal confirms specificity) .
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Use blocking peptides during Western blotting (e.g., 0.5 µg/mL antibody with/without immunogen peptide) to verify band disappearance .
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Validate cross-reactivity across human, mouse, and rat tissues via parallel IHC staining (e.g., brain tissue vs. epithelial systems) .
What experimental designs resolve contradictory TOCA-1 localization reports (junctional vs. cytoplasmic)?
Advanced analysis framework:
How to optimize TOCA-1 co-immunoprecipitation (Co-IP) conditions?
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Buffer optimization: Use mild detergents (1% Triton X-100) to preserve PDZ domain interactions critical for ZO-1 binding .
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Domain mapping: Precipitate ZO-1 truncation mutants (e.g., ΔPDZ1 vs. ΔPDZ3) to identify binding regions (PDZ1 domain required for interaction) .
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Controls: Include IgG-isotype controls and verify pull-down efficiency via immunoblotting for known partners (N-WASP, Diaph3) .
How does TOCA-1 antibody choice impact invadopodia quantification in metastasis models?
Technical considerations:
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Epitope selection: Antibodies targeting the N-terminus (aa 40–90) may miss isoforms lacking this region, while C-terminal antibodies (aa 450–467) detect full-length TOCA-1 .
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Functional validation: Correlate antibody signal with functional assays (e.g., Matrigel invasion in p53-silenced MTLn3 cells ).
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Multiplex staining: Combine with cortactin/phalloidin to distinguish invadopodia (degradation foci) from generic actin structures .
How to address batch variability in TOCA-1 immunohistochemistry?
Standardization protocol:
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Tissue pretreatment: Antigen retrieval using citrate buffer (pH 6.0) for FFPE sections .
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Titration curve: Test antibody concentrations from 2.5–20 µg/mL to optimize signal-to-noise in target tissues (e.g., human brain vs. breast tumors) .
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Inter-lot validation: Compare staining intensity across three independent antibody lots using a reference lysate (e.g., Human Brain Tissue Lysate ).
What computational tools analyze TOCA-1’s role in actin dynamics from live-cell imaging?
Quantitative workflow:
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Filopodia tracking: Use FIJI/ImageJ plugins (e.g., TrackMate) to measure protrusion rates in TOCA-1 KD vs. rescue models .
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Cross-correlation analysis: Calculate spatial-temporal alignment between TOCA-1, Ena, and F-actin in filopodia tips (≥0.7 Pearson coefficient indicates functional coupling) .
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Machine learning: Train U-Net models to segment TOCA-1 puncta in crowded junctional regions .
How to reconcile TOCA-1’s dual roles in suppressing metastasis (p53-dependent) and promoting invadopodia (p53-independent)?
Mechanistic dissection strategy:
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Transcriptional regulation: Perform ChIP-seq in p53-WT vs. mutant breast cancer cells to confirm TOCA-1 promoter binding .
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Post-translational modification: Screen for phosphorylation sites (e.g., Cdc42-binding domain) using Phos-tag gels.
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Pathway inhibition: Treat TOCA-1-high tumors with Cdc42 inhibitors (e.g., ML141) to assess metastatic suppression .
What controls are essential for TOCA-1 antibody reuse in longitudinal studies?
Quality assurance pipeline:
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Storage: Aliquot antibodies in PBS/0.02% azide; avoid >3 freeze-thaw cycles .
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Benchmarking: Include a reference sample (e.g., HEK293T overexpressing TOCA-1) in every experiment to monitor signal drift.
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Cross-validation: Alternate between Western blot (68 kDa band) and IF (junctional puncta) to confirm reagent stability .
