CNGC15 Antibody

What is CNGC15 and why is it important for plant research?

CNGC15 is a nuclear-localized calcium channel involved in mediating nuclear calcium oscillations that regulate root endosymbioses with arbuscular mycorrhiza and nitrogen-fixing bacteria . It plays a dual role in modulating root meristem development and nitrate-induced gene expression . CNGC15 functions as a homotetrameric complex and contains critical domains including the S1-S4 helices and C-linker region that are essential for channel gating . Given its significance in plant nutrition and development, antibodies against CNGC15 are valuable tools for investigating its expression, localization, interactions, and functional states across different tissues and conditions.

What structural features of CNGC15 are important to consider when selecting antibodies?

When selecting antibodies for CNGC15 research, consider these key structural features:

• The C-linker region, which is critical for channel gating and function

• The S1 helix containing conserved proline residues (e.g., P98/P104) where mutations create gain-of-function phenotypes

• The D408 residue located on the channel-facing side of helix A′, which forms important salt-bridge interactions with neighboring subunits

• The tetrameric assembly interface, as CNGC15 functions as a homotetramer

• Regions involved in cellular relocalization, as CNGC15 can move from the nuclear membrane to plasma membrane in columella cells under high nitrate conditions

Antibodies targeting these specific regions would allow researchers to distinguish between different functional states and conformations of CNGC15.

How do CNGC15 antibodies differ from antibodies for other CNGC family members?

CNGC15 antibodies must be carefully designed to avoid cross-reactivity with other CNGC family members, such as CNGC19 and CNGC20, which share structural similarities and can form heteromeric complexes . Key differences include:

• Specificity: While CNGC15 localizes primarily to the nuclear membrane, other CNGCs like CNGC20 are found predominantly at the plasma membrane

• Functional domains: Each CNGC has unique regions that can be targeted for specific antibody generation

• Post-translational modifications: Different CNGCs undergo distinct phosphorylation patterns - for instance, CNGC20 is phosphorylated and stabilized by BOTRYTIS INDUCED KINASE1 (BIK1)

• Expression patterns: CNGC15 is expressed in specific tissues and under particular conditions that differ from other family members

When selecting antibodies, researchers should verify specificity against multiple CNGC family members through appropriate controls and validation experiments.

What are the optimal sample preparation conditions for detecting CNGC15 in immunoblotting experiments?

For successful CNGC15 detection in immunoblotting, follow these guidelines:

For nuclear-specific extraction, additional nuclear isolation steps using sucrose or Percoll gradients may be necessary to enrich for CNGC15, given its primary nuclear localization .

How should researchers approach immunolocalization studies for CNGC15?

For accurate immunolocalization of CNGC15, consider this methodological approach:

• Fixation: Use 4% paraformaldehyde for 30-60 minutes to preserve protein structure while allowing antibody access

• Permeabilization: Include multiple permeabilization steps with 0.1-0.5% Triton X-100 to ensure antibody access to nuclear membranes

• Antigen retrieval: Consider citrate buffer (pH 6.0) heat-mediated retrieval if initial staining is unsuccessful

• Blocking: Use 3-5% BSA with 0.1% Tween-20 for 1-2 hours at room temperature

• Primary antibody: Incubate with anti-CNGC15 at 1:100-1:500 dilution overnight at 4°C

• Secondary antibody: Use fluorophore-conjugated antibodies appropriate for confocal microscopy

• Nuclear counterstain: Include DAPI (1:1000) to verify nuclear localization

• Imaging: Employ confocal microscopy with z-stacking to capture the three-dimensional distribution

Pay particular attention to CNGC15 localization changes under different experimental conditions, as research shows CNGC15 can relocalize from the nuclear membrane to the plasma membrane in columella cells upon high nitrate treatment .

What controls are essential when using CNGC15 antibodies in experimental procedures?

Implementation of proper controls is critical for CNGC15 antibody experiments:

The search results describe genotyping protocols for Atcngc15 mutants that can be adapted to generate appropriate control materials for antibody validation .

How can researchers use CNGC15 antibodies to study its role in nuclear calcium oscillations?

CNGC15 antibodies can be powerful tools for investigating nuclear calcium oscillations:

• Co-immunoprecipitation studies:

  • Use CNGC15 antibodies to pull down protein complexes

  • Identify interactions with DMI1, which functions as a pacemaker for calcium oscillations

  • Analyze different complex compositions under symbiotic vs. non-symbiotic conditions

• Calcium imaging correlation:

  • Perform immunofluorescence with CNGC15 antibodies

  • Combine with calcium sensors (e.g., GCaMP) in the same cells

  • Correlate CNGC15 localization intensity with calcium oscillation patterns

• Channel activity analysis:

  • Use conformation-specific antibodies to distinguish active vs. inactive channel states

  • Apply in fixed tissues at different timepoints during calcium oscillations

  • Quantify the proportion of active channels during different oscillation phases

• Mutant phenotype characterization:

  • Compare wildtype CNGC15 vs. autoactive mutants (P98S/P104S) with antibodies

  • Correlate protein levels with calcium oscillation frequency

  • Assess how mutations in the S1 helix (P98S/P104S) affect antibody binding and channel function

The search results indicate that CNGC15 generates nuclear calcium oscillations via a specific gating mechanism involving helix 1, while DMI1 acts as a pacemaker to regulate oscillation frequency .

What approaches allow researchers to investigate CNGC15 post-translational modifications?

Several approaches can be used to study CNGC15 post-translational modifications:

• Phosphorylation-specific antibodies:

  • Develop antibodies against predicted phosphorylation sites

  • Validate using phosphatase treatment controls

  • Apply in immunoblotting to detect changes in phosphorylation status

• Immunoprecipitation coupled with mass spectrometry:

  • Use CNGC15 antibodies for immunoprecipitation

  • Analyze precipitated proteins by mass spectrometry

  • Identify specific phosphorylation, ubiquitination, or other modifications

• In vitro kinase assays:

  • Based on protocols used for related CNGC20 phosphorylation by BIK1

  • Incubate immunoprecipitated CNGC15 with purified kinases

  • Detect phosphorylation using phospho-threonine antibodies

• Stability assays:

  • Adapt the cell-free protein stability assay described for CNGC20

  • Assess how post-translational modifications affect CNGC15 half-life

  • Compare wildtype and mutant CNGC15 stability under different conditions

The research on CNGC20 provides a valuable methodological template, as it demonstrates that BIK1 phosphorylates and stabilizes CNGC20, which could inform similar studies on CNGC15 .

How can CNGC15 antibodies help elucidate the functional differences between wildtype and mutant channels?

CNGC15 antibodies can provide critical insights into functional differences between wildtype and mutant channels:

• Conformational analysis:

  • Develop conformation-specific antibodies that distinguish between closed and open states

  • Compare epitope accessibility in wildtype vs. gain-of-function mutants (P98S/P104S)

  • Correlate with calcium imaging data to link structure to function

• Protein-protein interaction studies:

  • Use co-immunoprecipitation with CNGC15 antibodies

  • Compare interacting partners between wildtype and mutant CNGC15

  • Identify differential interactions that explain altered calcium oscillation frequencies

• Subcellular localization analysis:

  • Track CNGC15 distribution in different cell types using immunofluorescence

  • Compare wildtype localization with mutants under various treatments

  • Quantify nuclear vs. plasma membrane distribution in response to environmental cues

• Expression level quantification:

  • Use quantitative immunoblotting to measure protein levels

  • Assess if mutations affect protein stability or accumulation

  • Correlate expression with phenotypic outcomes in plant development and symbioses

The search results indicate that autoactive CNGC15 mutants generate spontaneous low-frequency calcium oscillations that enhance endosymbiotic relationships in plants .

How should researchers troubleshoot low signal-to-noise ratios when using CNGC15 antibodies?

When facing low signal-to-noise issues with CNGC15 antibodies:

• Antibody quality assessment:

  • Perform titration experiments to determine optimal concentration

  • Test fresh antibody aliquots to rule out degradation

  • Consider affinity purification against the immunizing antigen

• Sample preparation optimization:

  • Enrich for nuclear fractions to concentrate CNGC15

  • Modify extraction buffers to better preserve epitopes

  • Test multiple fixation protocols for immunofluorescence

• Signal amplification methods:

  • Implement tyramide signal amplification for immunofluorescence

  • Use high-sensitivity ECL substrates for immunoblotting

  • Consider biotin-streptavidin amplification systems

• Background reduction strategies:

  • Increase washing duration and stringency

  • Add 0.1-0.3% Triton X-100 to antibody dilution buffers

  • Pre-absorb antibodies with plant extract from CNGC15 knockout tissue

• Alternative detection approaches:

  • Compare antibody results with CNGC15-GFP fusion expression

  • Use epitope tagging with well-characterized tag antibodies

What strategies help distinguish between closely related CNGC family members?

To distinguish CNGC15 from related CNGC family members:

• Epitope selection:

  • Target unique sequences with minimal homology to other CNGCs

  • Focus on non-conserved regions outside the cyclic nucleotide-binding domain

  • Consider using peptides from the variable N- or C-terminal regions

• Validation approach:

  • Test antibodies on tissues from knockout lines of multiple CNGC family members

  • Perform peptide competition with peptides from related CNGCs

  • Use heterologous expression systems for specificity testing

• Localization distinction:

  • Exploit the nuclear localization of CNGC15 versus plasma membrane localization of other CNGCs

  • Use cellular fractionation followed by immunoblotting

  • Employ co-localization studies with known compartment markers

• Functional differentiation:

  • Utilize the unique role of CNGC15 in nuclear calcium oscillations

  • Compare antibody signals in symbiotic versus non-symbiotic conditions

  • Assess correlations with specific calcium oscillation frequencies

The research demonstrates that CNGC15 has distinct localization patterns compared to other family members like CNGC20, which primarily localizes to the plasma membrane .

How can researchers optimize CNGC15 antibody use for co-immunoprecipitation experiments?

For successful CNGC15 co-immunoprecipitation:

• Extraction buffer optimization:

  • Use mild non-ionic detergents (0.5% NP-40 or Digitonin)

  • Include stabilizing agents (10% glycerol, 1 mM DTT)

  • Add calcium chelators or calcium at physiological concentrations

• Cross-linking considerations:

  • For transient interactions, use DSP (dithiobis(succinimidyl propionate))

  • Apply membrane-permeable cross-linkers at low concentrations (0.5-2 mM)

  • Optimize cross-linking time (5-30 minutes) and quenching conditions

• Antibody coupling strategies:

  • Pre-couple antibodies to protein A/G beads to reduce heavy chain contamination

  • Use covalent coupling to beads using dimethyl pimelimidate

  • Consider oriented coupling using Protein A/G-conjugated magnetic beads

• Washing optimization:

  • Test gradient washing with decreasing salt concentrations

  • Include detergent in early washes, remove in later washes

  • Determine minimum number of washes that maintain specific interactions

• Elution methods:

  • Compare different elution conditions (low pH, high salt, peptide competition)

  • For mass spectrometry, use on-bead digestion to minimize contaminants

  • When blotting, use non-reducing conditions if studying multimeric complexes

This approach has been successfully applied to study protein interactions in plant immune signaling pathways involving related CNGC proteins .

How might CNGC15 antibodies help investigate the relationship between calcium oscillation frequency and downstream signaling?

CNGC15 antibodies could advance our understanding of calcium frequency-dependent signaling:

• Temporal dynamics analysis:

  • Use time-course immunoprecipitation to capture CNGC15 complexes during oscillations

  • Correlate complex composition with specific oscillation frequencies

  • Identify frequency-dependent interaction partners

• Conformational state mapping:

  • Develop antibodies recognizing distinct conformational states

  • Track the proportion of channels in each state during different oscillation patterns

  • Correlate with downstream gene expression changes

• Functional domain analysis:

  • Generate domain-specific antibodies to track exposure of regulatory regions

  • Investigate how mutations affecting oscillation frequency impact domain exposure

  • Correlate with changes in downstream phenotypes

The search results indicate that the frequency of calcium oscillations encodes specificity in symbiotic signaling - high frequency activates endosymbiosis programs, while low frequency modulates phenylpropanoid pathways .

What methods can help investigate CNGC15 assembly and trafficking using antibodies?

To study CNGC15 assembly and trafficking:

• Assembly dynamics:

  • Use non-denaturing immunoprecipitation to preserve complexes

  • Apply blue native PAGE to separate intact CNGC15 tetramers

  • Employ antibodies targeting subunit interfaces or assembled tetramers

• Trafficking pathway investigation:

  • Perform immunofluorescence with markers for ER, Golgi, and nuclear membrane

  • Track newly synthesized CNGC15 using pulse-chase approaches

  • Investigate how high nitrate treatment triggers relocalization to plasma membrane

• Interaction with trafficking machinery:

  • Immunoprecipitate CNGC15 and identify associated trafficking proteins

  • Use proximity labeling (BioID) coupled with CNGC15 antibodies

  • Analyze how mutations affect interactions with trafficking components

• Stimulus-dependent relocalization:

  • Quantify nuclear vs. plasma membrane distribution after treatments

  • Investigate the mechanisms of nitrate-induced relocalization in columella cells

  • Determine if relocalization correlates with changes in calcium signaling patterns

The research shows that CNGC15 can relocalize from the nucleus to plasma membrane in columella cells specifically upon high nitrate treatment, suggesting sophisticated trafficking regulation .

How can antibody-based approaches help translate CNGC15 research to agricultural applications?

Antibody-based approaches could facilitate agricultural applications of CNGC15 research:

• Biomarker development:

  • Use CNGC15 antibodies to assess channel activation in crop plants

  • Develop diagnostic kits to evaluate symbiotic potential in field conditions

  • Monitor CNGC15 status as an indicator of plant nutritional state

• Crop improvement screening:

  • Screen germplasm collections for beneficial CNGC15 variants

  • Identify cultivars with enhanced nuclear calcium signaling

  • Develop high-throughput immunoassays for breeding programs

• Symbiotic enhancement monitoring:

  • Track CNGC15 activation during interactions with beneficial microbes

  • Assess how agricultural practices affect CNGC15 function

  • Evaluate the effectiveness of biofertilizer applications

• Translational research:

  • Compare CNGC15 expression and function across crop species

  • Investigate if wheat CNGC15 mutations enhance nutrient acquisition

  • Develop antibodies specific to crop orthologs of CNGC15

Research demonstrates that autoactive CNGC15 enhances beneficial root endosymbioses in both model plants and wheat, increasing nutrient acquisition and reducing dependence on inorganic fertilizers .