COR413IM1 Antibody
Description
Target Protein Overview
COR413IM1 is an integral membrane protein localized to the chloroplast IEM, identified in Arabidopsis thaliana as part of the cold-regulated (COR) protein family . It plays roles in:
Antibody Development and Validation
The COR413IM1 antibody has been employed in multiple experimental systems to confirm protein localization and function:
Key Validation Methods
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Western Blotting: Detects COR413IM1–protein A fusion constructs (~30 kDa) in chloroplast envelope fractions .
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Subcellular Fractionation: Confirms IEM localization by resistance to trypsin digestion in intact chloroplasts .
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Immunogold Labeling: Validates envelope-specific targeting in transgenic plants .
Cross-Reactivity
The antibody shows specificity for COR413IM1-derived epitopes, including fusion partners like Staphylococcus protein A .
Chloroplast Protein Targeting Studies
COR413IM1 antibody enabled the validation of chimeric constructs designed to localize cyanobacterial bicarbonate transporters (e.g., BicA, SbtA) to the chloroplast IEM. Key findings include:
Cold Stress Responses
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COR413IM1 expression increases during cold acclimation, with the antibody quantifying its upregulation in Arabidopsis .
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T-DNA insertion mutants showed normal freezing tolerance, suggesting functional redundancy within the COR413IM family .
Membrane Protein Topology
Studies using the antibody demonstrated that COR413IM1's sixth transmembrane domain determines the orientation of fused passenger proteins (e.g., bicarbonate transporters) in the IEM .
Technical Considerations
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Limitations: Low expression levels of COR413IM1 fusion constructs in transgenic plants require sensitive detection methods .
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Controls: Purity of chloroplast fractions (e.g., Tic110 for IEM, LHCP for thylakoid markers) is critical for accurate localization .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
FAQs for COR413IM1 Antibody in Academic Research
How do I validate the specificity of COR413IM1 antibody for chloroplast inner envelope membrane (IEM) studies?
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Methodological Answer:
Perform Western blotting using chloroplast subfractions (stroma, thylakoid, envelope) isolated via sucrose density gradient centrifugation. Compare signals in envelope fractions against negative controls (e.g., stromal proteins like Rubisco). Validate specificity by:-
Detecting a ~30 kDa band corresponding to Cor413im1-protein A fusions in IEM fractions .
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Using trypsin protection assays: Intact chloroplasts treated with trypsin should retain Cor413im1 signals (IEM-protected), while OEM markers like Toc75 degrade .
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Include Arabidopsis knockout mutants (e.g., cor413im1 T-DNA lines) as negative controls .
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What protocols are recommended for detecting Cor413im1 in transgenic plants expressing chimeric bicarbonate transporters?
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Methodological Answer:
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SDS-PAGE: Use 12% or 5–20% gradient gels for optimal resolution of Cor413im1-containing chimeric proteins (e.g., BicAI, SbtAII) .
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Immunoblotting: Load 20–40 μg of total leaf protein, and probe with COR413IM1 antibody alongside protein A-tagged controls (e.g., Cor413im1-pA). Include TEV protease-treated samples to confirm cleavage of chimeric proteins (e.g., loss of ~30 kDa fragment) .
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Membrane fractionation: Isolate chloroplasts, treat with Triton X-100 or Na₂CO₃, and confirm Cor413im1 remains insoluble (integral membrane protein) .
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How can COR413IM1 antibody resolve contradictions in transporter topology at the chloroplast IEM?
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Methodological Answer:
Use protease accessibility assays combined with domain-specific tagging:-
Fuse HA or FLAG tags to cytosolic vs. stromal domains of chimeric transporters (e.g., BicA fused to full-length Cor413im1 vs. truncated K124).
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Treat isolated chloroplasts with trypsin and monitor tag degradation via Western blot.
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COR413IM1 antibody detects the IEM-anchored Cor413im1 portion, while anti-HA confirms transporter topology. Example:
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What experimental designs address low signal intensity of Cor413im1 in chimeric protein studies?
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Methodological Answer:
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Signal amplification: Use chemiluminescent substrates with extended exposure times (30 min–2 hr) for low-abundance targets .
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Cross-validation: Pair COR413IM1 antibody with protein A-specific probes (e.g., IgG-Fc binding) to confirm chimeric protein integrity .
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Quantitative controls: Include a reference protein (e.g., Tic110 for IEM) to normalize Cor413im1 signal intensity across samples .
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How to distinguish between authentic Cor413im1 and degradation products in Western blots?
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Methodological Answer:
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Degradation markers: Compare band patterns with transgenic lines expressing Cor413im1-pA (expected ~30 kDa) vs. degradation-prone constructs (e.g., SbtAI shows ~25 kDa fragments) .
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Protease inhibition: Include EDTA-free protease inhibitors during chloroplast isolation to minimize artifactual cleavage .
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Size calibration: Use prestained markers (e.g., 25–75 kDa range) to differentiate full-length proteins from truncated forms .
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What controls are critical when analyzing Cor413im1 in membrane protein interaction studies?
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Methodological Answer:
Control Purpose Expected Outcome Untransformed Arabidopsis Baseline signal No Cor413im1 bands OEM marker (Toc75) Validate fraction purity Degraded in trypsin-treated samples IEM marker (Tic110) Confirm IEM integrity Protected from trypsin Soluble protein (Rubisco) Rule out stromal contamination Absent in envelope fractions
Technical Notes
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Critical buffers: For alkaline extraction (0.1 M Na₂CO₃, pH 11.5), use fresh solutions to maintain pH efficacy .
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Topology studies: Combine COR413IM1 antibody with TEV protease cleavage assays to confirm transporter orientation (cleavage = stroma-facing) .
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Avoid pitfalls: Optimize antibody dilution (1:1,000–1:5,000) to reduce non-specific binding to chloroplast membrane proteins .
