At4g20460 Antibody
Description
Antibody Development and Validation
Antibodies against plant proteins are generated using recombinant or peptide antigens. Key validation steps include:
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Specificity Testing: Western blotting against wild-type and mutant plant lysates to confirm target recognition .
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Immunohistochemistry (IHC): Localization in plant tissues, often with epitope unmasking protocols for enhanced signal .
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Cross-Reactivity Checks: Ensuring no off-target binding to related proteins .
For example, in a study validating C9ORF72 antibodies, researchers used knockout (KO) mouse models to confirm specificity, a method applicable to plant antibody validation .
Research Applications of Plant-Specific Antibodies
While AT4G20460-specific studies are sparse, analogous workflows for plant antibodies include:
| Application | Method | Example Findings |
|---|---|---|
| Protein Localization | Fluorescent IHC | Subcellular targeting (e.g., Golgi, nucleus) |
| Expression Profiling | Western Blot | Tissue-specific expression patterns |
| Functional Studies | Co-Immunoprecipitation (Co-IP) | Identification of protein interaction partners |
Such approaches are critical for characterizing poorly studied plant proteins .
Challenges in Plant Antibody Development
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Low Protein Abundance: Plant proteins like AT4G20460 may require sensitive detection methods (e.g., luminescence-based assays) .
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Epitope Conservation: Cross-reactivity risks with homologous proteins in other plant species .
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Validation Resources: Limited availability of KO lines for specificity testing in plants .
Future Directions
The absence of direct data on the At4g20460 Antibody underscores the need for:
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Target Characterization: Functional annotation of AT4G20460 via CRISPR/Cas9-generated mutants.
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Antibody Production: Collaborative efforts to develop recombinant antibodies using phage display or hybridoma technologies .
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Multi-Omics Integration: Linking antibody-based protein data with transcriptomic or degradome datasets .
Product Specs
Composition: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
FAQs for At4g20460 Antibody in Academic Research
What are the primary research applications of At4g20460 antibody in plant cell wall biosynthesis studies?
The At4g20460 antibody is primarily used to investigate the role of UDP-d-Xyl 4-epimerase enzymes in Arabidopsis cell wall polysaccharide biosynthesis. Key applications include:
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Localization studies: Tracking the enzyme’s subcellular distribution (e.g., Golgi apparatus via GFP fusion assays) .
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Functional analysis: Validating enzyme activity in mutants (e.g., mur4 alleles with reduced l-Ara content) .
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Expression profiling: Correlating protein levels with developmental stages or stress conditions using Western blotting .
How can researchers validate the specificity of At4g20460 antibodies for immunoblotting?
Specificity validation requires a multi-step approach:
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Knockout controls: Compare protein detection in wild-type vs. At4g20460 T-DNA insertion mutants .
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Cross-reactivity checks: Test against homologs (e.g., At2g34850, At5g44480) due to high sequence similarity (>76% identity) .
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Orthogonal methods: Confirm results with RNAi knockdown followed by qRT-PCR and enzymatic activity assays .
What experimental designs are optimal for studying At4g20460’s role in Golgi-mediated polysaccharide modification?
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Brefeldin A treatment: Use 100 µg/mL for 2 hours to disrupt Golgi integrity and observe redistribution of At4g20460-GFP fusion proteins .
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Co-localization assays: Pair with markers like cis-Golgi MAN1 or trans-Golgi STtmd-mRFP .
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Enzymatic activity assays: Measure UDP-d-Xyl 4-epimerase activity in microsomal fractions isolated from mutant vs. wild-type plants .
How to investigate functional redundancy between At4g20460 and its homologs (At2g34850, At5g44480)?
How to resolve contradictions in At4g20460’s post-transcriptional regulation across studies?
Conflicting reports on miRNA-mediated regulation (e.g., degradome-seq vs. DMS-MaPseq data) require:
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Multi-omics integration: Cross-reference degradome-seq, small RNA-seq, and proteomics datasets .
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In vitro cleavage assays: Validate miRNA-At4g20460 interactions using 5′ RACE-PCR .
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Temporal profiling: Monitor protein/mRNA ratios across developmental stages to assess translational repression .
What strategies address data variability in At4g20460 localization studies?
Table 1: Genetic Variants of At4g20460 and Phenotypic Impacts
| Allele | Mutation Type | Amino Acid Change | Phenotype |
|---|---|---|---|
| mur4-1 | Exon 7 (Gly→Asp) | G275D | 50% reduced l-Ara in cell walls |
| mur4-3 | Exon 8 (Arg→Gln) | R304Q | Altered epimerase kinetics |
| mur4-4 | Intron 8 splice site | AG→AA | Mislocalization to ER-Golgi interface |
Table 2: miRNA Targeting At4g20460 (Degradome-seq Evidence)
| miRNA | Cleavage Site | 5′/3′ Arm | AGO Association | Functional Impact |
|---|---|---|---|---|
| Novel miRNA | 5p-2 / 3p-2 | 5′ shift | AGO2-enriched | Reduced protein abundance (~40%) |
