At5g45960 Antibody
Description
Definition and Target Protein
The At5g45960 Antibody recognizes the protein encoded by the At5g45960 gene in A. thaliana, a member of the GDSL esterase/lipase superfamily. This enzyme is implicated in plant defense mechanisms and lipid metabolism, with roles in pathogen-induced responses .
| Gene/Protein Information | Details |
|---|---|
| Gene Symbol | At5g45960 |
| Protein Name | GDSL esterase/lipase At5g45960 |
| Molecular Weight | 42,482 Da |
| UniProt Accession | Q9FJ40 |
| RefSeq ID | NM_123964.4 |
| Protein ID | NP_199408.1 |
Types and Characteristics
The antibody is available in two primary formats: monoclonal and polyclonal, each with distinct technical profiles.
Monoclonal Antibody (Mouse-Derived)
| Parameter | Value |
|---|---|
| Host | Mouse |
| Isotype | IgG |
| Clonality | Monoclonal |
| Immunogen | N-terminal synthetic peptide (Q9FJ40) |
| Applications | Western Blot (WB) with AbInsure™ validation at 1:1000 dilution |
| Specificity | GDSL esterase/lipase At5g45960 |
| ELISA Titer | ~10,000 (1 ng detection on WB) |
| Purity/Form | Lyophilized supernatant stored at -20°C |
| Source | Abmart (Package: X1-Q9FJ40 [ABX]) |
Polyclonal Antibody (Rabbit-Derived)
| Parameter | Value |
|---|---|
| Host | Rabbit |
| Isotype | IgG |
| Applications | ELISA, WB |
| Immunogen | Full-length recombinant At5g45960 protein |
| Purity/Form | Antigen-affinity purified, liquid with 0.03% Proclin 300 |
| Storage | -20°C or -80°C (avoid freeze-thaw cycles) |
| Source | MyBioSource (Catalog: MBS9011720) |
Applications in Research
The At5g45960 Antibody is primarily used in:
-
Western Blotting: Detects the protein in plant tissue lysates, validated at 1:1000 dilution .
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Enzyme-Linked Immunosorbent Assay (ELISA): Quantifies antigen levels in biological samples .
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Immunoprecipitation (IP): Isolates the target protein for downstream analysis .
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Plant Immunity Studies: Investigates the role of At5g45960 in pathogen response pathways .
Recombinant Protein and Antibody Validation
| Recombinant Protein | Details |
|---|---|
| Host Systems | E. coli, Yeast, Baculovirus, Mammalian Cells |
| Purity | ≥85% (SDS-PAGE) |
| Applications | ELISA, WB |
Antibody Performance Metrics
| Metric | Monoclonal | Polyclonal |
|---|---|---|
| Cross-Reactivity | Minimal (target-specific) | May vary by batch |
| Detection Limit | 1 ng (WB) | 0.01–1 ng (WB) |
| Recommended Dilution | 1:1000 (WB) | 1:500–1:1000 (WB) |
Role in Plant Immunity
Studies using the At5g45960 Antibody have identified its involvement in:
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Pathogen-Triggered Responses: Upregulated expression during viral infections (e.g., Turnip Mosaic Virus) .
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Lipid Metabolism: Potential role in modifying membrane lipids to restrict pathogen spread .
Comparative Analysis with Other Antibodies
| Feature | At5g45960 Antibody | Typical Polyclonal Antibodies |
|---|---|---|
| Specificity | High (N-terminal peptide) | Moderate to High |
| Affinity | ~10,000 (ELISA titer) | Variable |
| Batch Consistency | Monoclonal: High | Polyclonal: Moderate |
Challenges and Considerations
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Specificity Risks: Cross-reactivity with non-target proteins (e.g., AMPD2 or TRIM28) has been documented in other antibodies, highlighting the need for orthogonal validation .
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Storage and Handling: Lyophilized monoclonal antibodies require reconstitution and strict temperature control (-20°C) .
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Host-Species Reactivity: Limited to A. thaliana; cross-reactivity with other species not reported .
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
The following FAQs address key considerations for researchers working with the AT5G45960 antibody in academic settings, structured to reflect both foundational and advanced research challenges. Content is synthesized from available technical documentation and generalized antibody research methodologies .
Advanced Research Questions
How to resolve contradictory results between Western blot and qPCR data when studying AT5G45960 expression patterns?
Implement a tiered verification protocol:
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Technical Validation
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Confirm antibody batch consistency via parallel blotting of reference samples
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Verify RNA integrity (RIN >8) and cDNA synthesis efficiency
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-
Biological Validation
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Use orthogonal methods (e.g., CRISPR-Cas9 tagged lines with fluorescence reporters)
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Analyze temporal expression dynamics across diurnal cycles
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What strategies mitigate cross-reactivity concerns in non-model plant species?
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Perform in silico epitope mapping against homologous sequences in target species
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Combine antibody data with:
⇒ RNAi knockdown experiments
⇒ Mass spectrometry verification of immunoprecipitated proteins
Methodological Framework for Data Interpretation
Quantitative analysis workflow for AT5G45960 subcellular localization studies
| Step | Technique | Validation Metric |
|---|---|---|
| 1 | Confocal microscopy | Co-localization with organelle markers (Pearson’s r >0.7) |
| 2 | Fractionation WB | Enrichment in nuclear vs. cytoplasmic fractions |
| 3 | Functional assays | CRISPR mutants showing expected phenotypic deviations |
Contradiction Analysis Protocol
Discrepancy resolution matrix for conflicting antibody performance across studies
| Issue | Diagnostic Test | Resolution Pathway |
|---|---|---|
| Variable band patterns | Heat-denatured vs native PAGE | Re-evaluate protein extraction buffers |
| Seasonal expression differences | Controlled environment growth chambers | Standardize photoperiod/temperature |
| Tissue-specific epitope masking | Enzymatic pre-treatment (e.g., proteinase K) | Optimize antigen retrieval |
