CNGC16 Antibody
Description
CNGC16 Antibody: Functional Context
CNGC16 is a Ca²⁺-permeable cation channel activated by cyclic nucleotides (e.g., cGMP) and modulated by Ca²⁺/calmodulin . The antibody enables detection and analysis of CNGC16 expression, localization, and downstream signaling effects. Key applications include:
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Quantifying cGMP levels: Antibody-binding assays revealed heat stress increases 3′,5′-cyclic guanyl monophosphate (cGMP) in pollen and leaves, linking CNGC16 activity to stress responses .
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Pollen viability studies: Mutant pollen (cngc16-1 and cngc16-2) showed hypersensitivity to external CaCl₂, mirroring phenotypes in other CNGC mutants .
Stress-Dependent Pollen Fitness
Genetic disruptions of CNGC16 caused severe reductions in pollen viability under heat/drought stress, with rescue experiments confirming its necessity .
| Condition | Wild-Type Seed Set (%) | cngc16 Mutant Seed Set (%) | Rescue Efficiency (%) |
|---|---|---|---|
| No Stress | 73–75 | 69–73 | 100 |
| Hot/Cold Stress | 75 | 49–52 | 100 (via CNGC16 transgene) |
| 32°C Stress | 75 | 53 | 100 |
| 37°C Stress | 75 | 48–52 | 100 |
Data derived from segregation distortion assays under varying stress conditions .
Transcriptional Regulation
CNGC16 mediates heat-stress responses by activating heat shock transcription factors (HsfA2, HsfB1) and Bag6, which are critical for pollen germination :
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qPCR analysis: Mutants showed attenuated expression of these genes under stress.
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Proposed pathway: Heat → cGMP ↑ → CNGC16 activation → Ca²⁺ influx → transcriptional heat shock response .
Calcium Sensitivity
CNGC16 mutants exhibited hypersensitivity to external CaCl₂, suggesting disrupted Ca²⁺ homeostasis . This aligns with CNGCs’ role in integrating cyclic nucleotide and Ca²⁺ signals .
Functional Redundancy
CNGC16 operates independently of CNGC18 (essential for pollen tube growth), indicating specialized roles within the CNGC family .
Comparative Analysis of CNGCs in Stress Responses
| CNGC | Tissue Specificity | Stress Role | Mutant Phenotype |
|---|---|---|---|
| CNGC16 | Pollen | Heat/drought tolerance | 10-fold ↓ seed set under stress |
| CNGC6 | Vegetative tissues | Heat tolerance | Reduced thermotolerance |
| CNGC2 | Moss/Arabidopsis | Hyperactive stress response | Enhanced sensitivity at lower temps |
Adapted from studies comparing CNGC isoforms .
Technical Applications of CNGC16 Antibody
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Pollen-specific expression validation: Immunodetection confirmed CNGC16 localization in pollen .
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Rescue assays: Transgenic GFP-CNGC16 restored mutant phenotypes, verified via antibody-based tracking .
Unresolved Questions
Product Specs
Constituents: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Target Background
Q&A
How do researchers validate CNGC16 antibody specificity in pollen stress response studies?
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Method: Perform dual validation via (a) immunoblotting using Arabidopsis thaliana wild-type and cngc16 knockout pollen extracts, and (b) rescue assays with GFP-tagged CNGC16 transgenes under stress conditions .
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Key controls:
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Data interpretation: Reduced signal in knockouts and restored signal in rescued lines confirm specificity .
What experimental designs address conflicting reports on CNGC16’s role in calcium signaling versus transcriptional regulation?
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Contradiction: CNGC16 directly mediates Ca²⁺ influx in pollen tubes , yet also modulates heat shock transcription factors (e.g., HsfA2, HsfB1) .
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Resolution strategy:
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Combine live-cell Ca²⁺ imaging (e.g., GCaMP6f) with simultaneous qPCR of heat shock genes in cngc16 mutants .
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Use pharmacological blockers (e.g., LaCl₃ for Ca²⁺ channels) to decouple ionic and transcriptional effects .
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Employ cell-type-specific promoters (ACA9 for pollen tubes) in complementation assays .
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How to optimize CNGC16 antibody protocols for differentiating isoform-specific roles in stress adaptation?
What advanced techniques resolve CNGC16’s subcellular localization discrepancies under drought stress?
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Conflict: Some studies report plasma membrane localization, while others detect nuclear signals during heat stress .
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Methodology:
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Structured illumination microscopy (SIM): Achieves 100 nm resolution to distinguish membrane vs. proximal nuclear signals .
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Fractionation assays: Compare CNGC16 distribution in membrane/nuclear extracts under 37°C vs. 22°C .
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Bimolecular fluorescence complementation (BiFC): Test interactions with nuclear transporters (e.g., importin-α) .
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How to design genome-wide screens for CNGC16-interacting partners in pollen heat tolerance?
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Library: Use a pollen-specific yeast two-hybrid (Y2H) library from heat-stressed Arabidopsis .
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Selection criteria:
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Validation:
What statistical approaches reconcile CNGC16 mutant segregation ratios in stress assays?
| Genotype | Seed Set (Control) | Seed Set (37°C) | p-value |
|---|---|---|---|
| WT | 35.2 ± 2.1 | 32.8 ± 1.9 | >0.05 |
| cngc16-1 | 34.7 ± 1.8 | 3.1 ± 0.4 | <0.001 |
