At4g21770 Antibody
Description
Functional Role of At4g21770 Protein
The At4g21770 gene encodes a pseudouridine synthase involved in RNA modification, a critical process for chloroplast development and photosynthetic efficiency. Key findings include:
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Chloroplast Biogenesis: At4g21770 interacts with VAR2 (a chloroplast protease) to suppress leaf variegation phenotypes, indicating its role in maintaining chloroplast integrity .
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RNA Processing: As a pseudouridine synthase, it modifies ribosomal RNA (rRNA) to ensure proper chloroplast translation machinery .
3.1. Chloroplast Studies
At4g21770 Antibody has been pivotal in identifying the protein’s localization to chloroplasts. Studies using knockout mutants (svr1-1, svr1-2) revealed:
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Epistatic Interaction: Loss of At4g21770 function suppresses var2 mutant phenotypes, restoring chloroplast stability .
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Molecular Pathways: The protein participates in RNA quality control, preventing misfolded protein accumulation in chloroplasts .
3.2. Genetic Regulation
GWAS analyses linked At4g21770 SNPs to natural variation in photosynthetic light-use efficiency (ΦPSII), highlighting its role in acclimation to fluctuating light conditions :
| Gene | SNP Position | Phenotypic Association | Description |
|---|---|---|---|
| AT4G21770 | 11,561,583 | β-Glucosidase47 activity | Carbohydrate metabolic process |
4.1. Product Availability
Cusabio offers the antibody with guaranteed validation metrics:
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Cross-Reactivity: Specific to Arabidopsis thaliana isoforms.
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Batch Consistency: Rigorous quality control ensures reproducibility .
4.2. Case Studies
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Knockout Mutant Analysis: svr1-2 mutants showed no detectable At4g21770 transcripts, confirming the antibody’s specificity in null allele identification .
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Chloroplast Proteomics: Used to track At4g21770 expression under stress conditions, revealing upregulated activity during light stress .
Future Directions
Ongoing research aims to:
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Elucidate At4g21770’s role in RNA methylation and its impact on stress tolerance.
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Engineer Arabidopsis lines with modulated At4g21770 expression to enhance photosynthetic yield.
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How can researchers validate the specificity of At4g21770 Antibody in Western blot assays?
Specificity validation requires a multi-step approach:
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Recombinant Protein Control: Express and purify the full-length At4g21770 protein in a heterologous system (e.g., E. coli). Use this as a positive control to confirm the antibody detects the ~45 kDa band corresponding to At4g21770 .
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Knockout Mutant Lines: Compare protein extracts from wild-type Arabidopsis and at4g21770 T-DNA insertion mutants. The absence of the target band in mutants confirms specificity .
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Preabsorption Test: Preincubate the antibody with excess recombinant At4g21770 protein. Loss of signal in Western blot indicates specificity.
Table 1: Key Validation Parameters for At4g21770 Antibody
| Parameter | Experimental Setup | Expected Outcome |
|---|---|---|
| Recombinant Control | 1 µg purified protein, 1:1,000 dilution | Single band at ~45 kDa |
| Knockout Comparison | 20 µg total protein from WT/mutant extracts | Band present in WT, absent in mutant |
| Preabsorption | Antibody + 10x molar excess of antigen | Complete loss of signal |
What experimental conditions optimize At4g21770 Antibody performance in ELISA?
Optimal ELISA conditions depend on:
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Coating Concentration: Titrate recombinant At4g21770 protein from 0.1–5 µg/mL. Maximal signal-to-noise ratios are typically observed at 1 µg/mL .
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Blocking Buffer: Compare 5% BSA vs. non-fat milk. BSA reduces non-specific binding in plant extracts due to lower lipid content.
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Antibody Dilution: Test 1:500–1:5,000 dilutions. For CSB-PA871184XA01DOA, 1:2,000 in TBST + 1% BSA yields consistent results .
How should non-specific bands be addressed when using At4g21770 Antibody?
Non-specific bands arise due to cross-reactivity with homologous proteins or post-translational modifications. Mitigation strategies include:
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Gradient SDS-PAGE: Use 10–15% gels to improve resolution of closely migrating bands.
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Cross-Adsorption: Preadsorb the antibody with protein extracts from at4g21770 mutants to remove non-specific binders.
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Alternative Lysis Buffers: Replace RIPA buffer with Tris-HCl (pH 8.0) + 1% Triton X-100 to reduce protein aggregation.
How can epitope mapping clarify At4g21770 Antibody’s binding region?
Epitope mapping involves:
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Truncated Protein Constructs: Express overlapping fragments of At4g21770 (e.g., N-terminal 1–200 aa, C-terminal 201–410 aa) and test antibody reactivity. A study on SVR1 (a homolog) used similar methods to localize functional domains .
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Peptide Competition: Synthesize 15-mer peptides spanning the At4g21770 sequence. Preincubate the antibody with each peptide; loss of signal indicates epitope region.
Table 2: Epitope Mapping Workflow
| Step | Method | Outcome |
|---|---|---|
| Protein Fragmentation | Express 1–200 aa and 201–410 aa | Identifies reactive domain |
| Peptide Screening | 15-mer peptides, 10 µM each | Pinpoints epitope to 10 aa resolution |
What computational tools predict cross-reactivity risks for At4g21770 Antibody?
Advanced cross-reactivity analysis integrates:
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BLASTp Homology Screening: Identify proteins with >30% sequence similarity to At4g21770 (e.g., other pseudouridine synthases like SVR1 ).
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Structural Modeling: Use AlphaFold2 to compare 3D structures of At4g21770 and homologs. Regions with structural overlap may cause cross-reactivity.
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Machine Learning: Implement tools like Absolut! to simulate antibody-antigen binding landscapes and predict off-target interactions .
How do researchers reconcile contradictory data from At4g21770 Antibody across studies?
Contradictions often stem from variability in:
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Post-Translational Modifications (PTMs): Phosphorylation or ubiquitination may alter epitope accessibility. Treat samples with phosphatase inhibitors (e.g., PhosSTOP) or proteasome inhibitors (e.g., MG132) to stabilize PTMs.
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Tissue-Specific Expression: At4g21770 expression varies between roots and leaves. Normalize protein loads using organelle-specific markers (e.g., chloroplast Ponceau S staining) .
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Antigen Retrieval Methods: For fixed tissues, compare heat-induced (HIER) vs. enzymatic retrieval. HIER in citrate buffer (pH 6.0) improves signal in chloroplast-rich samples .
How can quantitative mass spectrometry (MS) validate At4g21770 Antibody specificity?
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Immunoprecipitation-MS: Immunoprecipitate At4g21770 from wild-type and mutant extracts. MS should detect At4g21770 exclusively in WT samples.
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Parallel Reaction Monitoring (PRM): Quantify unique peptides from At4g21770 (e.g., VLEDFGR and LQSNYLR). Correlation between antibody signal and peptide abundance confirms specificity .
