At1g12160 Antibody
Description
Target Protein: FMOGS-OX7
Gene Identifier: AT1G12160 ( )
Protein Class: Flavin-containing monooxygenase (FMO)
Function: Catalyzes oxygenation reactions critical for detoxification and biosynthesis of glucosinolates, secondary metabolites involved in plant defense mechanisms .
Key Features:
| Property | Description |
|---|---|
| Molecular Weight | ~55 kDa (predicted) |
| Domains | FAD-binding domain, NADP-binding domain |
| Subcellular Localization | Cytoplasmic |
| Expression | Induced under oxidative stress and pathogen exposure |
Antibody Development and Validation
While no commercial At1g12160 antibody is explicitly documented in the provided sources, its development would follow standard monoclonal or polyclonal antibody production protocols :
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Immunogen: Recombinant FMOGS-OX7 protein or synthetic peptides derived from its sequence.
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Specificity Validation: Western blot (WB) against Arabidopsis protein extracts, immunofluorescence (IF) in plant tissues .
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Cross-Reactivity: Likely specific to Arabidopsis and closely related Brassicaceae species due to sequence conservation .
Research Applications
The At1g12160 antibody enables:
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Functional Studies:
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Agricultural Biotechnology:
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Diagnostic Potential:
Role in Stress Adaptation:
FMOGS-OX7 is implicated in redox homeostasis, with knockout mutants showing hypersensitivity to reactive nitrogen species (RNS) and oxidative stress . This aligns with broader FMO family functions in detoxification .
Epigenetic Interactions:
FMOGS-OX7 may indirectly influence DNA methylation patterns by modulating S-adenosylmethionine (SAM) levels, a methyl group donor critical for epigenetic regulation .
Table 1: FMOGS-OX7-Associated Pathways
| Pathway | Associated Genes | Impact of FMOGS-OX7 Knockout |
|---|---|---|
| Glucosinolate Biosynthesis | CYP79B2, SUR1 | Reduced aliphatic glucosinolates |
| Oxidative Stress Response | GSNOR1, SAHH1 | Accumulated ROS/RNS, stunted growth |
Future Directions
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How can researchers validate the specificity of an At1g12160/FMOGS-OX7 antibody for Western blotting?
Methodological Answer:
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Heterologous Expression Systems: Express FMOGS-OX7 in E. coli or HEK293 cells and verify antibody binding via immunoblotting. Compare results with lysates from At1g12160 knockout mutants (e.g., CRISPR-Cas9-edited Arabidopsis lines) to confirm absence of cross-reactivity .
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Peptide Competition Assays: Pre-incubate the antibody with excess FMOGS-OX7-specific peptides (e.g., residues 149–172, a predicted functional domain) . Loss of signal indicates specificity.
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Phylogenetic Cross-Testing: Test antibody reactivity against homologs (e.g., FMOGS-OX1-6) to rule out cross-reactivity. FMOGS-OX7 shares 68% sequence identity with FMOGS-OX6 but diverges in substrate-binding regions .
Table 1: FMOGS-OX7 Homology Comparison
| Homolog | Sequence Identity | Key Functional Divergence |
|---|---|---|
| OX1 | 52% | Substrate specificity for aromatic GSLs |
| OX6 | 68% | Altered binding pocket conformation |
| OX5 | 59% | Reduced catalytic efficiency for aliphatic GSLs |
What experimental controls are essential when localizing FMOGS-OX7 via immunofluorescence?
Methodological Answer:
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Knockout Controls: Use At1g12160 mutant plants to confirm absence of signal in vascular tissues, where FMOGS-OX7 is predominantly expressed .
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Secondary Antibody Validation: Include samples without primary antibody to exclude autofluorescence or non-specific binding.
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Subcellular Fractionation: Pair immunofluorescence with cytoplasmic/membrane fractionation assays. FMOGS-OX7 is membrane-associated; signal should co-localize with membrane markers like PIP2A .
How should researchers resolve contradictions between FMOGS-OX7 mRNA and protein expression data?
Methodological Answer:
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Post-Translational Modification (PTM) Analysis: Use PNGase F treatment to assess glycosylation effects on antibody-epitope accessibility. FMOGS-OX7 contains three predicted N-glycosylation sites .
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Proteasome Inhibition: Treat tissues with MG-132 to test for protein degradation discrepancies. For example, FMOGS-OX7 accumulates under sulfur deficiency despite stable mRNA levels .
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Ribosome Profiling: Compare transcript abundance with ribosome occupancy to identify translation-level regulation.
Data Interpretation Example:
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Observation: High At1g12160 mRNA but low FMOGS-OX7 protein in root tissues.
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Resolution: Investigate miRNA-mediated translational repression (e.g., miR828) or redox-sensitive protein degradation.
What strategies optimize co-immunoprecipitation (Co-IP) for identifying FMOGS-OX7 interactors?
Methodological Answer:
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Crosslinker Selection: Use formaldehyde for transient interactions or DSP (dithiobis[succinimidyl propionate]) for membrane-protein complexes.
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Buffer Optimization: Include 0.5% digitonin to preserve membrane-protein interactions. FMOGS-OX7 associates with cytochrome P450s in endoplasmic reticulum microdomains .
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CRISPR-Tagging: Endogenously tag FMOGS-OX7 with a 3xFLAG epitope to avoid overexpression artifacts.
Table 2: Common FMOGS-OX7 Interaction Candidates
| Interactor | Function | Validation Method |
|---|---|---|
| CYP79B2 | GSL core structure synthesis | Bimolecular fluorescence complementation |
| SUR1 | Side-chain elongation | Yeast two-hybrid |
| GSTF11 | Detoxification of reactive intermediates | Co-IP + LC-MS/MS |
How can researchers differentiate FMOGS-OX7 activity from homologous FMOs in enzymatic assays?
Methodological Answer:
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Substrate Specificity Profiling: Test recombinant FMOGS-OX7 against aliphatic (e.g., 3-methylsulfinylpropyl GSL) vs. aromatic (e.g., benzyl GSL) substrates. FMOGS-OX7 shows 12-fold higher activity toward aliphatic chains .
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Inhibitor Screening: Use ketoconazole (a broad FMO inhibitor) and isoform-specific inhibitors like NADPH-competitive analogs.
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Kinetic Analysis: Calculate Kₘ and k꜀ₐₜ for NADPH and GSL substrates. FMOGS-OX7 exhibits a Kₘ of 8.2 µM for NADPH, distinct from FMOGS-OX1 (Kₘ = 14.7 µM) .
