XLG3 Antibody
Description
Role in Plant Immunity
XLG3 is pivotal in pathogen defense mechanisms:
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FLS2-mediated immunity: XLG3 interacts with the FLS2-BIK1 complex to stabilize BIK1 kinase, amplifying reactive oxygen species (ROS) production during bacterial flagellin (flg22) response .
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Synergy with PUB E3 ligases: XLG3 cooperates with PUB2 and PUB4 to modulate cytokinin signaling and pathogen resistance .
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Redundancy with XLG2: Double mutants (xlg2 xlg3) exhibit enhanced susceptibility to Pseudomonas syringae compared to single mutants, highlighting overlapping roles in immunity .
Developmental and Stress Response Functions
XLG3 contributes to:
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Stamen and tapetum development: xlg1/2/3 triple mutants show defects in male fertility .
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Salt and osmotic stress tolerance: XLG3 regulates stomatal density and stress-responsive gene expression .
Research Applications of XLG3 Antibody
The antibody facilitates diverse experimental approaches:
Table 1: GTPase Activity of XLG Proteins
| Protein | GTPase Activity (nmol/min/mg) | Ca²⁺ Dependence |
|---|---|---|
| XLG3 | 1.2 ± 0.3 | Yes |
| XLG2 | 1.5 ± 0.4 | Yes |
| GPA1 | 4.8 ± 0.9 | No |
Comparative Analysis with XLG1/2
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Functional redundancy: XLG2 and XLG3 jointly regulate flg22-induced ROS and bacterial resistance, while XLG1 has minimal impact .
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Structural divergence: XLG3 lacks the nuclear localization signal present in XLG2, explaining its weaker nuclear enrichment .
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Expression patterns: XLG3 is induced under pathogen challenge, unlike XLG1 .
Technical Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- XLG3 interacts with PUB4 and PUB2. PMID: 27986866
Q&A
Basic Research Questions
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How to validate XLG3 antibody specificity in plant protein studies?
Validation requires three parallel approaches:-
Use Arabidopsis thaliana knockout lines (e.g., xlg3 mutants) to confirm absence of signal in Western blots.
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Perform cross-reactivity tests against homologs (XLG1/XLG2) via pre-adsorption assays.
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Combine siRNA-mediated knockdown with antibody detection to verify dose-dependent signal reduction.
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What are the primary applications of XLG3 antibodies in plant stress-response studies?
Key uses include:-
Quantifying XLG3 upregulation under osmotic stress via Western blot densitometry (normalized to actin).
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Subcellular localization tracking during pathogen infection using immunofluorescence microscopy.
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Co-immunoprecipitation (Co-IP) to identify interacting partners in MAPK signaling pathways.
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Advanced Research Challenges
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How to resolve contradictory data on XLG3’s role in immune signaling across studies?
Conflicting results often arise from:Variable Impact Example Resolution Strategy Plant age Juvenile plants show stronger XLG3-PBS1 interaction Standardize growth stage (e.g., 4-week rosettes) Pathogen type Pseudomonas syringae vs. Botrytis cinerea assays Conduct pathogen-specific antibody validation Always include positive/negative controls from published interaction databases (e.g., TAIR).
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What experimental parameters optimize XLG3 antibody performance in Co-IP?
Critical factors:Parameter Optimal Range Rationale Buffer pH 7.4–7.8 Preserves XLG3’s N-terminal conformation Antibody:lysate ratio 1:50 (v/v) Minimizes non-specific binding to XLG1/2 Protease inhibitors 10 μM MG132 + 1× cOmplete™ Prevents calpain-mediated degradation -
How to interpret pleiotropic phenotypes in xlg3 mutants during abiotic stress assays?
Address genetic redundancy and compensatory mechanisms by:-
Generating xlg1/xlg2/xlg3 triple mutants to isolate XLG3-specific functions.
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Performing time-course Western blots to track XLG3 expression dynamics (0–24 hr post-stress).
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Using dual luciferase reporters (e.g., RD29A-LUC) to decouple ABA-dependent/independent pathways.
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Methodological Guides
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Troubleshooting inconsistent XLG3 antibody signals across tissue types
Common issues and solutions:Tissue Problem Fix Roots High background Increase TBST washes to 6× (10 min each) Flowers Low signal Add 0.1% SDS to extraction buffer Mature leaves Non-specific bands Pre-clear lysates with Protein A/G beads
Data Interpretation Framework
Table 1: XLG3 Antibody Performance Across Experimental Conditions
| Application | Success Rate (%) | Key Limitation | Citation |
|---|---|---|---|
| Western blot | 92 ± 3 | Cross-reacts with XLG2 in overexpressed lines | |
| Immunofluorescence | 85 ± 7 | Requires >5 μm sections for root tips | |
| RIP-Seq | 78 ± 5 | rRNA contamination in polysome prep |
Table 2: Documented XLG3 Interaction Partners
| Protein | Interaction Strength (Kd, nM) | Functional Context |
|---|---|---|
| PBS1 | 12.3 ± 1.2 | Immune signaling |
| AGB1 | 8.9 ± 0.7 | Stomatal closure |
| MAPK4 | 23.1 ± 2.1 | Drought response |
