NPF5.1 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
Here’s a structured FAQ collection for researchers studying NPF5.1 Antibody, incorporating experimental design, data analysis, and advanced research considerations based on peer-reviewed findings:
What is the functional role of NPF5.1 in ABA homeostasis?
NPF5.1 regulates abscisic acid (ABA) transport in the seed coat during Arabidopsis development. It mediates ABA uptake into maternal tissues, influencing the balance between active ABA and its inactive metabolite, dihydrophaseic acid (DPA). Loss-of-function npf5.1 mutants show a 3–4× increase in DPA and a slight increase in ABA content, suggesting its role in compartmentalizing ABA catabolism .
Methodological Insight:
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Use LC-MS/MS for quantification of ABA and DPA in seed coat/endosperm fractions.
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Compare mutants (e.g., npf5.1–1, npf5.1–2) with wild-type controls under standardized growth conditions .
How is NPF5.1 expression localized experimentally?
NPF5.1 is expressed in the seed coat, validated via:
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Promoter-GUS assays: Histochemical staining in developing seeds.
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qRT-PCR: Tissue-specific RNA extraction from seed coats vs. embryos .
Key Data:
| Tissue | NPF5.1 Expression (Relative to WT) | Technique Used |
|---|---|---|
| Seed Coat | High | GUS staining, qRT-PCR |
| Embryo | Undetectable | qRT-PCR |
What experimental models are used to study NPF5.1?
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Mutant Lines: T-DNA insertion mutants (npf5.1–1, npf5.1–2) with impaired ABA transport .
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Double Mutants: Cross npf5.1 with npf4.6 (embryo-specific ABA importer mutant) to study tissue-specific ABA dynamics .
How does NPF5.1 interact with ABA metabolic pathways under stress?
NPF5.1-mediated ABA transport affects catabolic enzyme activity. In npf5.1, elevated DPA implies increased CYP707A-family hydroxylase activity.
Experimental Design:
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Apply GA biosynthesis inhibitors (e.g., paclobutrazol) to uncouple ABA/GA crosstalk.
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Measure germination rates and hormone metabolites in combinatorial mutants .
Why does npf5.1 suppress paclobutrazol resistance in npf4.6 mutants?
NPF5.1 in maternal tissues compensates for embryonic ABA uptake defects in npf4.6.
Data Contradiction Analysis:
| Genotype | Paclobutrazol Germination Rate | ABA Sensitivity |
|---|---|---|
| Wild Type | 30% | High |
| npf4.6 | 80% | Low |
| npf4.6 npf5.1 | 45% | Moderate |
This suggests ABA redistribution between seed compartments is critical for germination phenotypes .
What computational tools predict antibody-antigen interactions for NPF5.1 studies?
While not directly applied to NPF5.1, methods like RosettaAntibodyDesign (RAbD) enable de novo antibody design by grafting CDR loops onto stable scaffolds. Key steps:
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Generate antigen-antibody structural models (e.g., using AlphaFold2).
Validation:
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SPR/BLI: Measure binding kinetics (KD ≤ 100 nM target).
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Thermal Shift Assays: Confirm structural stability (ΔTm < 5°C) .
Technical Challenges and Solutions
Challenge: Discrepancy between ABA and DPA levels in npf5.1.
Resolution: Use compartment-specific metabolite profiling (e.g., laser-capture microdissection) to resolve spatial hormone dynamics .
Challenge: Antibody cross-reactivity in plant tissues.
Resolution: Validate antibodies via knockout-validated immunoblotting and epitope tagging (e.g., GFP-NPF5.1 fusions) .
