CAC2 Antibody
Description
Definition and Target
CAC2 Antibody is a polyclonal antibody developed against the biotin carboxylase subunit of heteromeric acetyl-coenzyme A carboxylase (ACCase) in Arabidopsis thaliana. ACCase catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, a critical step in fatty acid biosynthesis .
Key Features:
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Target Protein: Biotin carboxylase subunit (encoded by the CAC2 gene) .
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Molecular Weight: Recognizes a 51 kDa polypeptide in Arabidopsis extracts .
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Specificity: Reacts exclusively with the CAC2 subunit; preabsorption with the immunizing antigen abolishes reactivity .
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Functional Inhibition: Inhibits ACCase enzymatic activity in vitro, confirming its specificity .
Role in Fatty Acid Synthesis
CAC2 is essential for fatty acid biosynthesis in plants. The antibody has been used to:
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Study light-/dark-regulated ACCase activity in chloroplasts .
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Investigate tissue-specific expression patterns, with highest activity in organs synthesizing membrane lipids or oils (e.g., seeds, flowers) .
Gene Expression Coordination
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CAC2 expression mirrors that of CAC1 (encoding the biotin carboxyl carrier subunit), suggesting transcriptional coordination .
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Promoter analysis reveals shared regulatory motifs between CAC1 and CAC2, potentially governing co-expression .
Table 1: CAC2 Antibody Characteristics
| Property | Detail |
|---|---|
| Target Species | Arabidopsis thaliana |
| Immunogen | Glutathione S-transferase-CAC2 fusion protein |
| Applications | Western blot, enzymatic inhibition assays |
| Molecular Weight Detection | 51 kDa (native protein); 537 amino acids (precursor with transit peptide) |
| Regulatory Role | pH-dependent activity modulation in chloroplasts |
Table 2: CAC2 Expression Across Tissues
| Tissue | Expression Level | Functional Context |
|---|---|---|
| Seeds | High | Oil deposition |
| Flowers | High | Membrane lipid synthesis |
| Mature Leaves | Moderate | Baseline fatty acid production |
| Roots | Low | Limited metabolic demand for fatty acids |
Significance in Plant Biology
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Metabolic Regulation: CAC2-mediated ACCase activity is critical for balancing fatty acid synthesis under varying light conditions .
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Biotechnological Potential: Insights from CAC2 studies inform genetic engineering of oilseed crops to enhance lipid yields .
Limitations and Future Directions
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Current research is limited to model plants (A. thaliana); applicability in crops requires validation.
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Further studies could explore CAC2’s interaction with other ACCase subunits and metabolic regulators.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
Generating a collection of FAQs for "CAC2 Antibody" research requires synthesizing antibody development methodologies and experimental design principles from available academic sources. Below is a structured framework based on antibody research best practices and technical considerations from multispecific antibody development literature , combined with intent-driven question design strategies from search behavior analysis .
What validation methods are critical for confirming CAC2 antibody specificity in immunohistochemistry (IHC)?
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Methodological answer:
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Perform blocking peptide assays by pre-incubating the antibody with excess target peptide to confirm signal loss.
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Use genetic knockout controls (e.g., CRISPR/Cas9-modified cell lines lacking CAC2) to validate target binding .
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Cross-validate with orthogonal techniques like Western blotting or SPR (surface plasmon resonance) to assess binding kinetics.
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| Validation Step | Purpose | Acceptance Criteria |
|---|---|---|
| Peptide Blocking | Confirm epitope specificity | ≥80% signal reduction |
| Knockout Control | Rule off-target binding | No detectable signal |
| Cross-reactivity Screen | Assess family protein binding | ≤5% cross-reactivity |
How should researchers address contradictory data between ELISA and flow cytometry results for CAC2?
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Resolution framework:
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Buffer optimization: Adjust detergent concentrations (e.g., Triton X-100) to expose epitopes in flow cytometry .
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Conformational analysis: Use circular dichroism to determine if fixation alters CAC2’s tertiary structure.
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Epitope mapping: Employ hydrogen-deuterium exchange mass spectrometry to identify antibody binding regions affected by assay conditions.
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What strategies improve CAC2 antibody performance in multiplexed spatial proteomics workflows?
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Technical recommendations:
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Optimize antibody clonality: Use rabbit monoclonal antibodies (e.g., clone RB-9214-P0) for higher affinity (KD ≤ 1 nM) .
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Implement signal amplification systems: Tyramide-based amplification for low-abundance targets.
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Validate with reference datasets: Compare results against single-cell RNA sequencing profiles of CAC2-expressing tissues.
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| Parameter | Requirement | Tool/Resource |
|---|---|---|
| Affinity | KD ≤ 1 nM | BioLayer Interferometry |
| Multiplex Compatibility | No cross-talk with ≥5-plex panels | CODEX® validation |
| Batch Consistency | CV ≤ 15% across lots | ISO 13485 standards |
How can researchers resolve discrepancies in CAC2 subcellular localization across published studies?
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Analytical approach:
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Contextual metadata analysis: Compare cell lines/tissue sources used in conflicting studies (e.g., neoplastic vs. normal samples).
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Post-translational modification screening: Test for phosphorylation-dependent epitope masking using λ-phosphatase treatment.
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Single-molecule localization microscopy: Resolve ≤40 nm localization precision using dSTORM or PALM techniques .
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Methodological Guidance Table
| Research Stage | Key Challenge | Recommended Protocol | Validation Metrics |
|---|---|---|---|
| Target Validation | Off-target binding | CRISPR knockout + MS/MS proteomics | ≥10-fold signal reduction |
| Assay Development | Epitope accessibility | Antigen retrieval optimization (pH 9.0 citrate buffer) | CV ≤ 20% across runs |
| Data Interpretation | Context-dependent variability | Integrated omics correlation analysis | R² ≥ 0.7 vs. transcriptomics |
