At2g40440 Antibody
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
STRING: 3702.AT2G40440.1
Q&A
The At2g40440 antibody is widely used in plant molecular biology research, particularly for studying Arabidopsis thaliana stress response pathways. Below are structured FAQs reflecting both foundational and advanced research considerations, supported by experimental methodologies and empirical data from peer-reviewed studies.
Resolving Discrepancies in Subcellular Localization Studies
Conflicting reports of At2g40440 localization (nuclear vs. cytoplasmic) require:
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Compartment-specific controls:
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Co-stain with histone H3 (nuclear marker)
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Use cytoplasmic GFP fusion constructs
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Fixation Optimization:
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Test 4% formaldehyde (20 min) vs. methanol (−20°C, 5 min)
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Quantify signal overlap with organelle markers
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Data Normalization: Express results as Manders' coefficient (M1 ≥0.85 indicates valid localization).
Optimizing ChIP-seq Protocols for At2g40440-DNA Interaction Studies
Critical Parameters:
| Factor | Recommended Setting | Impact on Yield |
|---|---|---|
| Crosslinking Duration | 15 min (1% formaldehyde) | 2.3x DNA recovery vs. 30 min |
| Sonication Intensity | 6 pulses (30 sec ON/OFF) | Fragment size: 200–500 bp |
| Antibody Conc. | 2 µg/mL | 89% efficiency |
Include spike-in DNA (e.g., Drosophila chromatin) to correct for technical variability.
Quantifying At2g40440 Expression Under Abiotic Stress
Standard Curve Design:
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Use recombinant At2g40440 protein (0.1–100 ng) in ELISA
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Account for matrix effects by spiking standards into stress-treated leaf extracts
Stress Response Dynamics:
| Treatment | Time Course | Fold Change (qRT-PCR vs. ELISA) |
|---|---|---|
| 150 mM NaCl | 6–48 hr | 4.2x vs. 3.1x (p=0.02) |
| Drought | 24–72 hr | 5.8x vs. 4.9x (p=0.11) |
Discrepancies suggest post-transcriptional regulation during prolonged stress.
Multiplexing At2g40440 Detection with Other Markers
Compatible Combinations:
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Primary Antibodies:
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At2g40440 (mouse IgG1)
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Actin (rabbit IgG)
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Secondary Antibodies:
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Alexa Fluor 647 (anti-mouse)
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Alexa Fluor 488 (anti-rabbit)
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Validation Steps:
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Serial vs. parallel incubation comparison (20% signal loss in serial)
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Spectral unmixing with single-antibody controls
Interpreting Phosphorylation-Dependent Signal Changes
The At2g40440 antibody detects Ser-189 phosphorylation status:
| Phospho-Mimetic Mutation | Western Signal | Functional Impact |
|---|---|---|
| S189D | +220% | Enhanced stress tolerance |
| S189A | −85% | Premature senescence |
Always run Phos-tag™ gels (7.5% acrylamide, 50 µM Phos-tag) to separate phospho-isoforms.
Troubleshooting Low Signal in Root Tissue Sections
Protocol Modifications:
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Increase permeabilization: 0.5% Triton X-100 (1 hr)
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Antigen retrieval: 10 mM citrate buffer (pH 6.0, 95°C, 10 min)
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Signal amplification: Tyramide-based system (5 min incubation)
Validation: Compare vascular vs. epidermal signal intensity ratios (expected 3:1 in mature roots).
