GLN1-3 Antibody
Description
Target and Biological Role
GLN1-3 is one of several cytosolic glutamine synthetase (GS1) isoforms in plants, with distinct roles in nitrogen assimilation and remobilization. It is critical for:
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Ammonium assimilation in leaves and roots under varying nitrogen conditions .
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Kernel development in maize, where mutations reduce grain size and number .
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Functional redundancy with other GS1 isoforms (e.g., GLN1-1, GLN1-2) to maintain nitrogen homeostasis .
Table 1: Comparison of GLN1 Antibodies
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Cross-reactivity: Many GLN1 antibodies recognize multiple isoforms due to high sequence homology (e.g., 98% identity between GLN1-3 and GLN1-4 in maize) .
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Validation: Western blot bands at ~42 kDa confirm specificity .
3.1. Functional Studies in Plants
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Knockout Mutants: In Arabidopsis, triple gln1;1-gln1;2-gln1;3 mutants show:
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Maize Mutants: gln1-3 mutants exhibit:
3.2. Mechanistic Insights
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Promoter Analysis: GLN1-3 expression in maize is driven by tissue-specific promoters (e.g., RbcS for bundle sheath cells) .
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Enzyme Kinetics: GLN1-3 has lower substrate affinity than GLN1-1, suggesting specialized roles under high ammonium .
Technical Considerations
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Dilution Ranges:
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Buffer Compatibility: Antibodies are typically stable in PBS with 0.02 M potassium phosphate and 0.15 M NaCl .
Key Findings from Peer-Reviewed Studies
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Nitrogen Remobilization: GLN1-3 is essential for transferring nitrogen from leaves to seeds, with triple mutants showing 40–60% reductions in nitrogen flux .
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Circadian Regulation: In Arabidopsis, GLN1.3 expression is directly regulated by the circadian clock gene CCA1 .
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Biomass Impact: GS1 mutations do not affect shoot biomass, indicating a specific role in reproductive development .
Product Specs
Target Background
Q&A
Here’s a structured FAQ collection for researchers studying GLN1-3 antibodies, derived from peer-reviewed studies and technical documentation:
How do I validate the specificity of a GLN1-3 antibody in Western blot assays?
Methodological Answer:
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Perform peptide competition assays: Pre-incubate the antibody with the immunogenic peptide (e.g., KLH-conjugated synthetic peptide matching GLN1-3 epitopes) to confirm signal loss .
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Use knockout mutants (e.g., gln1-3 or gln1-3/gln1-4 double mutants) to verify absence of target bands .
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Compare results across tissues (e.g., roots vs. leaves) to confirm isoform-specific detection, as GLN1-3 expression varies spatially .
Supporting Data:
| Sample Type | Expected Band (kDa) | Observed in Wild Type | Observed in gln1-3 Mutant |
|---|---|---|---|
| Leaf extract | 40 | Yes | No |
| Root extract | 38 (GSr isoform) | Yes | Yes |
| Source: |
What controls are essential for GLN1-3 antibody experiments in plant tissues?
Methodological Answer:
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Positive controls: Wild-type tissue with confirmed GLN1-3 expression (e.g., non-senescent Arabidopsis leaves) .
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Negative controls:
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Cross-reactivity checks: Test phylogenetically distant species (e.g., Chlamydomonas algae) to assess antibody specificity .
How do I resolve contradictions in GLN1-3 expression data across studies?
Methodological Answer:
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Variable factors:
Example Workflow:
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Standardize growth conditions (N-source, light cycle).
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Use RT-qPCR to validate transcript levels alongside protein assays .
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Include CCA1-ox overexpressor lines to test regulatory redundancy .
What experimental designs address functional redundancy among GLN1 isoforms?
Methodological Answer:
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Multi-mutant analysis: Generate gln1;1/gln1;2/gln1;3 triple knockouts to unmask residual GS activity from minor isoforms .
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Complementation assays: Express GLN1-3 under tissue-specific promoters in mutants to isolate physiological roles .
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Metabolite profiling: Compare amino acid pools (e.g., Asn, Gln) in mutants under varying N regimes to infer isoform contributions .
Key Findings:
How do I optimize GLN1-3 antibody use for non-model species?
Methodological Answer:
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Phylogenetic alignment: Compare target species’ GLN1-3 sequence to immunogen peptide (e.g., Phaseolus vulgaris epitope) .
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Pre-absorption test: Incubate antibody with heterologous protein extracts (e.g., Zea mays) to reduce cross-reactivity .
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2D gel validation: Resolve isoforms by pI/mass differences (e.g., 40 kDa GS1-3 vs. 38 kDa GSr in maize) .
Why does my GLN1-3 antibody detect multiple bands in Western blots?
Methodological Answer:
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Post-translational modifications: Treat samples with phosphatases/proteases to rule out phosphorylation or degradation.
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Isoform cross-reactivity: Use gln1-3/gln1-4 double mutants to distinguish GS1-3 (absent) from GS1-4 (persists) .
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Buffer optimization: Increase SDS concentration (10%) and include 1 M Tris-HCl (pH 6.8) to improve denaturation .
