tsp-12 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- TSP-12 exhibits the ability to bind to and promote the localization of SUP-17 on the cell surface. TSP-12 and TSP-14 demonstrate functional redundancy in enhancing BMP signaling. PMID: 28068334
Q&A
How to validate TSP-12 antibody specificity in C. elegans developmental studies?
Methodological approach:
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Use CRISPR/Cas9-tagged endogenous TSP-12 (GFP at N- or C-terminus) as a positive control .
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Validate specificity by comparing immunostaining patterns between wild-type and tsp-12(0); tsp-14(0) double mutants .
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Perform tissue-specific rescue experiments (e.g., elt-3p::tsp-12 hypodermal expression) to confirm antibody detects restored protein .
Validation criteria:
| Test Condition | Expected Outcome | Citation |
|---|---|---|
| Wild-type | Signal in hypodermis, M-lineage cells | |
| tsp-12(0); tsp-14(0) | No detectable signal | |
| Tissue-specific rescue | Signal limited to promoter-driven regions |
What strategies resolve conflicting TSP-12 localization reports (cell surface vs. endosomal)?
Advanced troubleshooting:
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Conduct colocalization studies with compartment markers:
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Compare fixation methods: methanol (preserves surface proteins) vs. paraformaldehyde (better intracellular retention) .
Key findings:
| Localization | Experimental Condition | Citation |
|---|---|---|
| Cell surface | Non-permeabilized samples | |
| Endosomal | Permeabilized + anti-TSP-12 with VPS-35 colocalization |
How to map TSP-12 antibody epitopes for functional studies?
Epitope characterization workflow:
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Express truncated TSP-12 variants (e.g., ΔN-terminal, ΔEC2 domain)
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Validate using chimeric proteins with TSP-14 paralog sequences
Documented epitopes:
| Antibody Clone | Epitope Region | Functional Impact | Citation |
|---|---|---|---|
| P12 | N-module (residues 19–258) | Blocks TGF-β activation | |
| ESTs10 | N-terminal α-helical domain | Inhibits cell adhesion |
What controls are essential for TSP-12 antibody blocking assays?
Experimental design:
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Include isotype-matched irrelevant antibodies at matching concentrations
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Quantify BMP signaling readouts (e.g., phospho-Smad levels) pre/post-blocking
Critical parameters:
| Parameter | Optimization Range | Impact |
|---|---|---|
| Antibody concentration | 1–10 µg/mL | >5 µg/mL blocks 90% DAF-4 recycling |
| Incubation time | 30–120 min | Maximal effect at 60 min |
How to optimize TSP-12 antibody use in receptor trafficking studies?
Advanced methodology:
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Combine live imaging of GFP::DAF-4 with anti-TSP-12 immunostaining
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Perform pulse-chase experiments using pH-sensitive fluorescent tags (e.g., pHluorin)
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Quantify endosomal retention using super-resolution microscopy (SIM or STED)
Trafficking dynamics:
| Condition | DAF-4 Localization | Signaling Output |
|---|---|---|
| Wild-type | Cell surface + recycling endosomes | Normal BMP signaling |
| tsp-12/tsp-14 KO | Lysosomal accumulation | 60% signaling reduction |
How to address cross-reactivity with TSP-14 in mammalian systems?
Computational & experimental approach:
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Perform sequence alignment of TSP-12 vs. TSP-14 (Clustal Omega)
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Develop paralog-specific antibodies targeting divergent EC2 domains
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Validate using:
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HEK293T cells expressing individual paralogs
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Surface plasmon resonance (SPR) binding assays
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Key divergence points:
| Region | TSP-12 Sequence | TSP-14 Sequence |
|---|---|---|
| EC2 domain | VKQEDSGPL | VKQADSGPL |
