CNGC14 Antibody
Description
Definition and Biological Context of CNGC14 Antibody
The CNGC14 antibody is a specialized immunological tool designed to detect and study the Cyclic Nucleotide-Gated Channel 14 (CNGC14), a calcium-permeable ion channel critical for auxin signaling, root gravitropism, and cellular pH regulation in Arabidopsis thaliana. This antibody enables researchers to localize CNGC14 protein expression, assess its interaction partners, and investigate its role in rapid auxin responses, such as calcium influx, membrane depolarization, and apoplastic alkalinization .
Validation Data
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Specificity: Recognizes CNGC14 in root epidermal cells, particularly enriched in the transition zone plasma membrane .
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Mutant Controls: Absence of signal in cngc14 loss-of-function mutants confirms specificity .
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Functional Assays: Correlated with delayed auxin-induced growth inhibition and calcium transients in cngc14 mutants .
Role in Auxin Signaling
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Calcium Influx: CNGC14 mediates auxin-induced cytosolic Ca²⁺ elevation within 1 minute, essential for rapid root growth inhibition .
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Apoplastic Alkalinization: The antibody confirmed that CNGC14 is required for auxin-triggered root surface pH increases, a process delayed in cngc14 mutants .
Gravistimulation Responses
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CNGC14-dependent calcium signaling is critical for establishing pH gradients across gravistimulated roots. The antibody revealed diminished alkaline domains on the lower root side in cngc14 mutants, correlating with delayed bending .
Protein Interaction Studies
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AFB1 Receptor: CNGC14 acts downstream of the AFB1 auxin receptor. Immunostaining showed colocalization of AFB1-mCitrine and CNGC14-GFP in root epidermal cells .
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ARO Proteins: Structural modeling (AlphaFold3) predicted ARO2 tetramers stabilizing CNGC14 at the plasma membrane, validated via FRET-FLIM and colocalization assays .
Key Research Insights Using CNGC14 Antibody
Technical Considerations and Limitations
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Low Protein Abundance: CNGC14 accumulates at low levels in root tips, necessitating sensitive detection methods .
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Functional Redundancy: Partial functional overlap with other CNGCs (e.g., CNGC5/6/9) complicates phenotypic analysis .
Future Directions
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- BES1/BZR1 Degradation: Understanding Distinct Developmental and Environmental Cues in Plants PMID: 28286297
- CYCLIC NUCLEOTIDE-GATED CHANNEL 14 (CNGC14) plays a crucial role in the initial stages of auxin-induced ion signaling and growth inhibition within Arabidopsis roots. Mutations in cngc14 lead to the complete absence of rapid Ca(2+) and pH signaling in response to auxin treatment. PMID: 26752079
Q&A
Experimental Design for CNGC14 Antibody Validation
Question: How can I validate the specificity of a CNGC14 antibody in plant tissues? Answer: To validate the specificity of a CNGC14 antibody, you should perform experiments using both positive and negative controls. For example, use a mutant lacking CNGC14 or a line overexpressing CNGC14 to assess the antibody's specificity. Additionally, omitting the primary antibody or using a secondary antibody alone can help confirm the absence of non-specific binding. Include these controls in your experimental design to ensure reliable results .
Data Interpretation and Contradiction Analysis
Question: What are some potential contradictions in interpreting data from CNGC14 antibody experiments, and how can they be addressed? Answer: Potential contradictions may arise from differences in antibody specificity or experimental conditions. For instance, if the antibody recognizes multiple paralogs, this could lead to inconsistent results. Address these issues by:
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Specificity Testing: Use mutants or overexpression lines to confirm specificity.
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Experimental Replication: Repeat experiments under different conditions to ensure consistency.
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Data Normalization: Normalize data to account for variations in experimental setups .
Advanced Research Questions: Role of CNGC14 in Plant Physiology
Question: How does CNGC14 contribute to plant physiological processes, such as auxin signaling and gravitropism? Answer: CNGC14 plays a crucial role in auxin-induced calcium signaling and growth inhibition in plant roots. It mediates rapid calcium influx in response to auxin, which is essential for early growth inhibition and gravitropic responses. Mutants lacking CNGC14 exhibit delayed growth inhibition and impaired gravitropic bending .
Methodological Considerations for CNGC14 Studies
Question: What are some key methodological considerations when studying CNGC14 using antibodies? Answer: Key considerations include:
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Antibody Specificity: Ensure the antibody specifically recognizes CNGC14 and not other proteins.
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Tissue Preparation: Optimize tissue fixation and permeabilization to maintain protein integrity.
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Imaging Techniques: Use high-resolution microscopy to accurately localize CNGC14 in plant tissues .
Interactions with Other Proteins
Question: How does CNGC14 interact with other proteins, such as Armadillo Repeat Only (ARO) proteins? Answer: CNGC14 interacts with ARO proteins, which modulate its activity. Structural modeling suggests that AROs form a complex with CNGC14, potentially regulating its function in plant cells. These interactions are crucial for understanding the broader regulatory mechanisms involving CNGC14 .
Advanced Techniques for Studying CNGC14
Question: What advanced techniques can be used to study CNGC14 function and interactions? Answer: Techniques such as:
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Structural Modeling: Use tools like AlphaFold to predict protein structures and interactions.
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Microfluidics and Imaging: Employ microfluidics for dynamic imaging and membrane potential measurements.
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Genetic Screens: Conduct fluorescence-based genetic screens to identify mutants with altered CNGC14 function .
Data Analysis and Visualization
Question: How can I effectively analyze and visualize data from CNGC14 antibody experiments? Answer: Use statistical software to analyze data, focusing on quantitative measures of fluorescence intensity or protein localization. Visualize results using heat maps, bar graphs, or confocal microscopy images to clearly illustrate protein distribution and changes in response to treatments .
Future Research Directions
Question: What are some potential future research directions involving CNGC14? Answer: Future studies could explore:
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Mechanistic Insights: Investigate the detailed mechanisms of CNGC14 regulation by ARO proteins.
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Physiological Roles: Examine CNGC14's role in other plant physiological processes beyond auxin signaling.
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Cross-Species Comparisons: Compare CNGC14 function across different plant species to identify conserved and divergent aspects .
Data Table Example: CNGC14 Antibody Validation
| Experimental Condition | Fluorescence Intensity (AU) | Specificity Control |
|---|---|---|
| Wild-Type (WT) | 100 | Positive |
| CNGC14 Mutant | 20 | Negative |
| CNGC14 Overexpressor | 200 | Positive |
| No Primary Antibody | 0 | Negative |
