GLR3.4 Antibody
Description
Introduction to GLRAntibody
GLR3.4 Antibody is a polyclonal antibody developed against the Arabidopsis thaliana GLR3.4 protein, a member of the plant glutamate receptor-like (GLR) family. These receptors are homologs of animal ionotropic glutamate receptors (iGluRs) but exhibit distinct structural and functional features . The antibody enables detection and localization of GLR3.4 in experimental settings, facilitating research into its roles in plant signaling and development .
Functional Insights into GLR3.4
GLR3.4 is implicated in diverse physiological processes:
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Calcium Signaling: GLR3.4 forms heteromeric channels (e.g., with GLR3.2) that regulate Ca²⁺ influx, influencing lateral root primordia production .
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Pollen Tube Growth: Localizes to pollen tube tips, modulating cell polarity and Ca²⁺ gradients critical for growth .
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Stress Responses: Activated by amino acids (e.g., asparagine) and glutathione (GSH), with S-glutathionylation at cysteine C205 enhancing channel activity .
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Structural Features: Shares a three-layer domain architecture with iGluRs but exhibits unique symmetry, ligand-binding interfaces, and GSH-dependent gating mechanisms .
Research Applications of GLRAntibody
Key studies utilizing this antibody have revealed:
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Subcellular Localization: Plasma membrane localization in root phloem cells and pollen tubes .
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Protein Interactions: GLR3.4 forms functional heteromers with GLR3.2, as shown by genetic knockout phenotypes and electrophysiological assays .
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Ligand Binding: Structural studies resolved GSH binding to the amino-terminal domain (ATD), influencing channel activation .
Implications for Plant Biology
GLR3.4 Antibody has advanced understanding of:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- GLR3.4-mediated Ca2+ influx may be involved in the regulation of seed germination under salt stress. PMID: 29432559
- GLR3.4, along with other glutamate receptors (GLRs), could play a crucial role in the rapid transmission of environmental stress signals through Ca(2+) -based mechanisms. PMID: 15864638
- Research has explored the effects of 6 amino acids on the glutamate receptors of A. thaliana, highlighting the roles of GLR3.3 and GLR3.4 in the channel mechanisms. PMID: 18162597
Q&A
How to validate GLR3.4 antibody specificity in heterologous expression systems?
Methodological Answer:
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Co-expression with CNIH proteins: GLR3.4 requires CORNICHON HOMOLOG (CNIH1/CNIH4) for functional trafficking to the plasma membrane. Validate antibody specificity by co-expressing GLR3.4 with CNIH1/CNIH4 in COS-7 cells and confirming channel activation via electrophysiological responses to glutamate (Glu) or asparagine (Asn) .
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Calcium imaging: Measure cytosolic Ca²⁺ influx using fluorescent indicators (e.g., Fluo-4) upon ligand application. Wild-type GLR3.4 should show Ca²⁺ spikes, while mutants (e.g., C205A) exhibit reduced responses .
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Negative controls: Include cells expressing CNIH proteins alone or GLR3.4 mutants lacking critical residues (e.g., C205A) .
What physiological roles of GLR3.4 require antibody-based detection?
Advanced Research Context:
GLR3.4 regulates:
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Pollen tube tip-focused Ca²⁺ gradients: Use immunofluorescence to localize GLR3.4 at pollen tube tips and correlate with Ca²⁺ flux measurements .
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Root phloem signaling: Validate tissue-specific expression in Arabidopsis roots via immunolocalization or promoter-GUS fusions .
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Wound response pathways: Combine antibody staining with transcriptional reporters (e.g., JA-responsive LOX2) to link GLR3.4 activation to jasmonate signaling .
How to address non-specific binding in GLR3.4 antibody assays?
Optimization Strategies:
How to resolve contradictions in GLR3.4 ligand activation data?
Data Conflict Analysis:
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Expression system variability: Heterologous systems (e.g., COS-7 vs. Xenopus oocytes) may exhibit differing ligand sensitivities. Always include CNIH co-expression and validate with Ca²⁺ imaging .
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Post-translational modifications: S-glutathionylation at C205 potentiates GLR3.4 activity. Compare wild-type and C205A mutants using reducing vs. non-reducing SDS-PAGE .
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Temporal resolution: Use rapid perfusion systems to distinguish fast (Glu/Asn) vs. slow (GSH) activation kinetics .
How to detect phosphorylation-dependent regulation of GLR3.4?
Advanced Methodology:
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In vitro kinase assays: Incubate purified GLR3.4 cytoplasmic domains with calcium-dependent protein kinases (CDPKs) and ATP-γ-S, followed by Phos-tag gel electrophoresis .
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Bimolecular fluorescence complementation (BiFC): Co-express GLR3.7 (homolog) with 14-3-3ω in protoplasts to map interaction sites (e.g., Ser-860) .
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Phospho-specific antibodies: Generate custom antibodies against phosphorylated serine residues (e.g., pS860) and validate via peptide competition assays .
Designing a flow cytometry panel for GLR3.4-expressing cells
Experimental Design Framework:
How to study tissue-specific GLR3.4 localization?
Advanced Techniques:
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Immunogold electron microscopy: Localize GLR3.4 at subcellular compartments (e.g., pollen tube apical membrane) .
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Cell-type-specific promoters: Drive GLR3.4-GFP fusions using AtGLR3.4 native promoters in transgenic lines .
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Single-cell RNA-seq: Cross-validate antibody data with transcriptomic profiles from guard cells or root phloem .
What structural features impact GLR3.4 antibody epitope accessibility?
Key Considerations:
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ATD-LBD interactions: The clamshell-shaped amino-terminal domain (ATD) binds glutathione (GSH), potentially occluding epitopes. Use reducing agents (e.g., DTT) during immunoprecipitation .
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Transmembrane topology: Target extracellular epitopes (e.g., LBD loops) for live-cell staining without permeabilization .
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Post-fixation artifacts: Compare fresh vs. paraformaldehyde-fixed samples to assess epitope stability .
