FAD7 Antibody
Description
Introduction to FAD7 Antibody
The FAD7 antibody is a specialized immunological tool developed to detect and study the FAD7 protein, a plastid-localized ω3 fatty acid desaturase critical for synthesizing trienoic fatty acids (TAs) in plants . This enzyme introduces double bonds into fatty acid chains, influencing membrane fluidity and stress responses . The antibody targets specific epitopes on FAD7, enabling researchers to investigate its expression, localization, and functional roles in plant physiology .
Development and Specificity of FAD7 Antibody
-
Antigen Design: The antibody was raised against a synthetic oligopeptide corresponding to residues 69–84 of the mature Glycine max (soybean) FAD7 protein . This region is conserved across FAD7 isoforms but distinct from related desaturases (e.g., FAD3, FAD8), ensuring specificity .
-
Validation: Western blot analyses confirmed reactivity with a ~39–42 kDa band in soybean chloroplasts, correlating with FAD7’s theoretical molecular weight . Immunodepletion experiments using the immunogenic peptide abolished signal detection, verifying specificity .
Protein Localization Studies
-
Subcellular Localization: Immunofluorescence and immunogold labeling revealed FAD7’s preferential localization in chloroplast thylakoid membranes, with minor presence in the chloroplast envelope .
-
Tissue-Specific Expression: The antibody detected FAD7 in leaves, roots, and developing seeds, with post-translational modifications altering protein conformation across tissues .
Functional and Mechanistic Insights
-
Redox Sensitivity: In vitro experiments demonstrated that FAD7 protein conformations shift under varying redox conditions. For example, oxidizing agents like diamide induced a 42 kDa band, while reducing agents stabilized a 39 kDa form .
-
Gene Family Analysis: The antibody identified two FAD7 isoforms (GmFAD7-1 and GmFAD7-2) in soybean, resolving their differential expression patterns .
Role in Stress Responses
-
FAD7 abundance correlates with cold and pathogen stress tolerance, as TA levels influence membrane stability and signaling .
-
In Arabidopsis, FAD7 and its homolog FAD8 exhibit overlapping but distinct roles, with FAD7 constitutively expressed and FAD8 induced under stress .
Technical Considerations
-
Western Blot Optimization: Extended electrophoresis and urea-containing gels are required to resolve FAD7’s doublet bands .
-
Artifact Prevention: Thiol-reducing agents (e.g., β-mercaptoethanol) must be included in extraction buffers to avoid artificial aggregation .
Future Directions
Current research leverages FAD7 antibodies to:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Chloroplast omega-3 fatty acid desaturase (FAD7) is an enzyme involved in the biosynthesis of 16:3 and 18:3 fatty acids. These fatty acids are essential components of plant membranes, playing a vital role in maintaining membrane fluidity and function. FAD7 introduces the third double bond in the biosynthesis of these fatty acids, a critical step in their production. The enzyme is believed to use ferredoxin as an electron donor and acts on fatty acids esterified to galactolipids, sulfolipids, and phosphatidylglycerol.
- Studies have shown that cold-induced tobacco seedlings expressing FAD7 exhibit significantly enhanced survival rates compared to wild-type seedlings when exposed to low temperatures (0.5, 2 or 3.5 degrees C). This suggests that FAD7 plays a crucial role in cold tolerance. PMID: 16292565
- Further research has demonstrated that the ozone-induced expression of the FAD7 gene is dependent on salicylic acid (SA) but not on ethylene, jasmonic acid (JA), NPR1, or SID2. This finding highlights the specific regulatory pathways involved in FAD7 gene expression. PMID: 16415067
- FAD7 has been shown to catalyze the production of jasmonic acid, a plant hormone involved in various stress responses. Notably, FAD7 expression is locally induced by wounding. Additionally, RNA polymerase II C-terminal domain phosphatases have been identified as negative regulators of FAD7 expression, suggesting a complex interplay of regulatory mechanisms. PMID: 18764923
Q&A
Basic Research Questions
How to validate FAD7 antibody specificity in plant systems?
-
Perform sequential validation using:
-
Immunodepletion assays: Preincubate antibodies with target peptide antigen (e.g., VASIEEEQKSVDLTNG epitope) to confirm signal loss in Western blot/immunofluorescence .
-
Cross-species reactivity tests: Validate using species with known FAD7 sequence divergence (e.g., Medicago vs. soybean) .
-
Subcellular localization consistency: Compare antibody signals with chloroplast markers (e.g., chlorophyll autofluorescence) in immunofluorescence assays .
-
What are optimal methods to detect FAD7 isoforms in Western blot?
-
Use extended SDS-PAGE (12% acrylamide + 4M urea) with prolonged migration to resolve doublet bands (~39-42 kDa) caused by:
| Issue | Solution | Reference |
|---|---|---|
| Smearing | Increase urea concentration to 6M | |
| Weak signal | Use chemiluminescent peroxidase detection (e.g., SuperSignal West Pico) |
Advanced Research Questions
How to resolve contradictions in FAD7 subcellular localization data?
-
Multi-method validation:
-
Contextual interpretation: Consider developmental stage-specific localization (e.g., grana association in mature leaves vs. envelope preference in young tissues) .
Designing FAD7 functional studies across plant species: Key considerations
-
Epitope conservation analysis: Align target sequences using Clustal Omega; require ≥75% homology in antibody-binding regions .
-
Activity correlation: Pair antibody detection with GC-MS fatty acid profiling (e.g., 18:3/16:3 ratio as ω3 desaturase activity index) .
Interpreting tissue-specific FAD7 protein conformations
-
Methodological framework:
| Tissue | Observed Bands | Proposed Explanation |
|---|---|---|
| Mature leaves | Doublet (39/42 kDa) | GmFAD7-1 and -2 isoforms |
| Roots | Single band | Tissue-specific PTM suppression |
Methodological Best Practices
Quantitative imaging strategies for FAD7 localization studies
-
Use confocal line-scan analysis with dual-channel detection:
-
Channel 1: Anti-FAD7 (488 nm excitation)
-
Channel 2: Chlorophyll autofluorescence (640 nm)
-
-
Apply Pearson’s correlation coefficient threshold >0.85 for valid chloroplast localization .
