GONST1 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research suggests that GONST1 acts as a Golgi GDP-sugar transporter, specifically providing GDP-Man to the Golgi lumen for the synthesis of glycosylinositol phosphoceramides (GIPCs). PMID: 23695979
Q&A
Basic Research Questions
How to validate the specificity of GONST1 antibodies for Arabidopsis studies?
To confirm specificity, perform Western blotting using:
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Arabidopsis thaliana wild-type and gonst1 knockout mutants.
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Subcellular fractionation (e.g., Golgi-enriched P3 membranes vs. ER-rich P2 fractions) .
| Sample | Band Observed (kD) | Mutant Detection |
|---|---|---|
| Wild-type | 37 | Yes |
| gonst1 mutant | Absent | No |
| Yeast vrg4-2 + GONST1 | 37 | Yes |
Include peptide competition assays with the immunogen (e.g., N-terminal 14-amino-acid peptide) .
What methods confirm Golgi localization of GONST1 in plant cells?
Use immunofluorescence colocalization with Golgi markers (e.g., GFP-tagged Golgi residents) and transient expression in protoplasts. For quantitative data:
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Compare signal distribution in gonst1 mutants vs. wild-type .
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Validate using anti-Anp1p (yeast Golgi marker) in complemented yeast strains .
Advanced Research Questions
How to reconcile GONST1’s ability to transport multiple GDP-sugars in vitro with its in vivo specificity for GDP-mannose?
Experimental design considerations:
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Liposome assays: Test selectivity across GDP-Man, GDP-Glc, GDP-Fuc, and GDP-l-Gal under varying luminal conditions (e.g., pH, antiporter requirements) .
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Genetic complementation: Compare phenotypic rescue in yeast vrg4-2 mutants using GONST1 vs. other GDP-sugar transporters .
| Substrate | Transport Activity (nmol/min/mg protein) | gonst1 Phenotype Rescue |
|---|---|---|
| GDP-Man | 9.8 ± 1.2 | Full |
| GDP-Glc | 8.5 ± 0.9 | Partial |
| GDP-Fuc | 7.1 ± 0.7 | None |
Data from suggest in vivo specificity arises from substrate competition or regulatory interactions.
How does GONST1 deficiency affect glycosphingolipid mannosylation without altering cell wall polysaccharides?
Methodological approach:
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LC-MS/MS profiling: Compare glycosylinositol phosphoceramide (GIPC) composition in gonst1 vs. wild-type.
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Radiolabeling: Track -mannose incorporation into GIPCs and glucomannan .
| Component | Wild-Type Mannose (%) | gonst1 Mannose (%) |
|---|---|---|
| GIPCs | 12.3 ± 1.5 | 3.1 ± 0.8 |
| Glucomannan | 45.2 ± 2.7 | 44.9 ± 3.1 |
Findings indicate GONST1 preferentially supplies GDP-Man for sphingolipid biosynthesis .
How to optimize GONST1 antibody-based flow cytometry for B-cell screening in cross-reactive antibody studies?
Leverage dual-expression vectors and membrane-bound Ig display:
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Clone paired heavy/light chains into Golden Gate-compatible vectors for surface display .
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Use Alexa647/Alexa568-labeled probes (e.g., HA antigens) for multi-color sorting .
| Probe Combination | Antigen-Specific B Cells Identified |
|---|---|
| PR8 (H1N1) | 204 |
| H2 (H2N2) | 99 |
| PR8 + H2 | 71 |
This system enables rapid isolation of broadly reactive antibodies within 7 days .
What controls are critical when analyzing GONST1 expression via promoter-GUS fusions?
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Tissue-specific controls: Compare staining in vascular vs. non-vascular tissues (e.g., callus vs. leaves) .
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Timed staining: Limit substrate exposure to 30–60 minutes to avoid false positives in highly expressing tissues .
Data Contradiction Analysis
Why does GONST1 knockdown not alter glucomannan levels despite its in vitro GDP-Man transport activity?
Hypothesis testing:
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Compensatory transporters: Screen for paralogs (e.g., GONST2-4) via RNAi knockdown combinatorial lines.
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Subcellular trafficking: Use brefeldin A treatments to disrupt Golgi function and measure substrate accumulation .
These studies highlight the necessity of in vivo validation even for biochemically characterized transporters.
