DOF2.4 Antibody
Description
Antibody Validation Pipeline
High-quality antibodies require rigorous validation. A standardized workflow includes:
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Proteomics-driven cell line selection: Identifying cell lines with high target expression (e.g., using PaxDB) .
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CRISPR/Cas9 knockout (KO) controls: Generating isogenic KO lines to confirm antibody specificity .
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Multi-application screening: Testing antibodies in immunoblot, immunoprecipitation (IP), and immunofluorescence (IF) .
For DOF2, validation might involve:
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Immunoblot: Comparing parental and KO Arabidopsis lines to confirm loss of signal .
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Epitope mapping: Ensuring the antibody recognizes the conserved zinc finger domain .
Applications in Plant Research
DOF antibodies enable functional studies, including:
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Chromatin immunoprecipitation (ChIP): Mapping DNA-binding sites of DOF2 in gene promoters .
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Subcellular localization: Confirming nuclear localization via IF .
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Mutant phenotyping: Assessing developmental defects in DOF2 knockdown lines .
Example data from Arabidopsis antibody screens :
| Antibody Target | Cross-Reactivity Tested | Specificity Confirmed |
|---|---|---|
| DOF11 | 94 other proteins | Yes (no cross-reactivity) |
| MYB6 | Other MYB family members | Yes |
Challenges and Solutions
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Low target abundance: DOF transcription factors are often expressed at low levels, necessitating sensitive detection methods (e.g., chemiluminescence) .
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Sequence similarity: DOF family members share conserved domains, requiring careful epitope selection to avoid cross-reactivity .
Future Directions
Advances in antibody engineering, such as cell-free synthesis and site-specific conjugation (e.g., CRISPR-edited sortase tags), could improve the reproducibility of plant antibody production . For DOF2.4, further studies might:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- AtDof2.4 and AtDof5.8 may play roles in distinct, yet early, stages of vascular development. PMID: 17583520
Q&A
Basic Research Questions
What validation strategies ensure DOF2.4 antibody specificity for immunoblotting?
To confirm specificity:
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Perform genetic validation using CRISPR/Cas9-generated knockout (KO) cell lines. Compare immunoblot signals between parental and KO lysates (Figure 2 in ).
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Use orthogonal validation by correlating antibody signals with proteomics databases (e.g., PaxDB) for expected expression levels .
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Include loading controls (e.g., β-actin) and validate via immunoprecipitation followed by mass spectrometry .
How to optimize DOF2.4 antibody protocols for immunohistochemistry (IHC) versus immunocytochemistry (ICC)?
What controls are essential for DOF2.4 antibody experiments?
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Negative controls: KO cell lines, isotype-matched irrelevant antibodies.
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Positive controls: Cell lines with confirmed high DOF2.4 expression (validated via proteomics databases) .
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Technical controls: Secondary-only and no-primary-antibody conditions to rule out non-specific binding .
Advanced Research Questions
How to resolve discrepancies in DOF2.4 localization data across studies?
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Hypothesis 1: Epitope masking due to post-translational modifications. Test alternative epitope retrieval methods (e.g., enzymatic vs. heat-mediated) .
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Hypothesis 2: Antibody cross-reactivity. Perform immunodepletion assays by pre-incubating the antibody with recombinant DOF2.4 protein. Loss of signal confirms specificity .
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Hypothesis 3: Cell-type-specific splicing variants. Validate using isoform-specific KO models .
What methodologies enable quantitative analysis of DOF2.4 expression levels?
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Dual-labeling assays: Combine DOF2.4 antibody with a reference protein antibody (e.g., GAPDH) for normalization.
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Standard curve approach: Use recombinant DOF2.4 protein serial dilutions to establish a linear signal range (Table 3 in ).
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Digital quantification: Software-based analysis (e.g., ImageJ) for Western blot band intensity or fluorescent signal quantification .
How to design multiplex assays using DOF2.4 antibody without cross-talk?
Methodological Notes
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For low-abundance targets, use recombinant multiclonal antibodies to enhance sensitivity while maintaining specificity .
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In flow cytometry, prioritize indirect detection with signal-amplifying secondary antibodies (e.g., biotin-streptavidin systems) .
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Always validate DOF2.4 antibody performance in native vs. denatured conditions, as epitope accessibility varies with sample processing (Table 2 in ).
