NCED9 Antibody

What is NCED9 and why is it important in plant research?

NCED9 is one of nine NCED family members in Arabidopsis thaliana, specifically involved in ABA biosynthesis during seed development. It catalyzes the oxidative cleavage of 9-cis-epoxycarotenoids to xanthoxin, which is considered the key regulatory step in ABA biosynthesis . NCED9 is expressed in both embryo and endosperm tissue during mid-development stages, and genetic studies have shown that it works in conjunction with other NCEDs (particularly NCED6) to regulate seed dormancy through ABA production . Unlike NCED3, which primarily functions in stress responses, NCED9's role is more focused on developmental processes .

What are the typical applications of NCED9 antibodies in plant research?

NCED9 antibodies are valuable tools for:

• Protein localization studies via immunohistochemistry and immunofluorescence

• Quantification of NCED9 protein levels via western blotting

• Immunoprecipitation for studying protein-protein interactions

• Chromatin immunoprecipitation (ChIP) assays to study transcriptional regulation

• ELISA-based quantitative analysis of NCED9 expression

These applications help researchers investigate NCED9's role in ABA biosynthesis, seed development, and stress responses in plants .

How do I determine the specificity of an NCED9 antibody?

Determining antibody specificity is critical for accurate results. The following methodological approach is recommended:

• Positive and negative controls: Use tissue samples from wild-type plants versus nced9 mutants .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application.

• Cross-reactivity testing: Test against recombinant proteins of other NCED family members.

• Western blot validation: Verify single band detection at the expected molecular weight (~65 kDa for NCED9).

• Knockout/knockdown validation: Compare signals between wild-type and NCED9-deficient samples .

Additional validation could include mass spectrometry confirmation of immunoprecipitated proteins to ensure specificity against other NCED family members.

How can NCED9 antibodies be used to investigate the spatiotemporal regulation of ABA biosynthesis in seeds?

Methodological approach:

• Tissue-specific immunolocalization:

  • Fix seed tissues at different developmental stages using 4% paraformaldehyde

  • Perform microtome sectioning (5-10 μm thickness)

  • Apply NCED9 primary antibody (1:200-1:500 dilution)

  • Use fluorescent or peroxidase-conjugated secondary antibodies

  • Counterstain with DAPI for nuclear visualization

  • Compare with in situ hybridization and promoter-reporter fusion patterns

• Co-localization studies:

  • Double-immunostaining with antibodies against other ABA biosynthesis enzymes

  • Combine with chloroplast markers (NCED9 is chloroplastic)

  • Correlate with tissue-specific expression data from transcriptome analyses

• Developmental profiling:

  • Systematically sample seeds at defined developmental stages (early, mid, late)

  • Perform protein extraction with tissue-specific extraction protocols

  • Quantify NCED9 protein levels via quantitative western blotting

  • Compare protein levels with transcriptional activity patterns

This approach allows correlation between NCED9 localization/abundance and ABA levels across seed development.

What techniques can overcome cross-reactivity issues between NCED family members when using antibodies?

The NCED family in Arabidopsis has nine members with potential structural similarities that may cause cross-reactivity issues. To address this:

• Epitope selection strategy:

  • Target unique regions of NCED9 (N- or C-terminal regions) rather than conserved domains

  • Perform detailed sequence alignment of all nine NCED family members

  • Select peptide immunogens with minimal homology to other NCEDs

  • Consider using recombinant protein fragments rather than synthetic peptides

• Validation protocols:

  • Test antibodies against recombinant proteins of all NCED family members

  • Use knockout mutants for each NCED as negative controls

  • Perform immunoprecipitation followed by mass spectrometry to identify any cross-reactive proteins

  • Preabsorb antibodies with recombinant proteins of other NCED family members

• Alternative approaches:

  • Consider epitope tagging of NCED9 in transgenic plants when direct antibodies show cross-reactivity

  • Use highly purified monoclonal antibodies rather than polyclonal antibodies

  • Implement proximity ligation assays for enhanced specificity

The table below outlines key regions of NCED proteins that might help in generating specific antibodies:

How can I quantitatively assess NCED9 protein levels in different plant tissues and under various stress conditions?

For quantitative assessment of NCED9 protein:

• Protein extraction optimization:

  • Use specific buffers for different tissues (seeds require stronger extraction conditions)

  • Include protease inhibitors and reducing agents

  • Consider subcellular fractionation (NCED9 is chloroplastic)

  • Optimize protein extraction from seeds at different developmental stages

• Quantitative western blotting:

  • Use internal loading controls (actin, tubulin, or GAPDH)

  • Include calibration curves with recombinant NCED9 protein

  • Employ fluorescence-based secondary antibodies for wider dynamic range

  • Use digital imaging and analysis software for quantification

• ELISA development:

  • Sandwich ELISA with capture and detection antibodies

  • Competitive ELISA with NCED9 protein standards

  • Validation across different tissue types and conditions

• Data normalization and analysis:

  • Normalize protein levels to total protein content

  • Compare with transcript levels via RT-qPCR

  • Correlate with ABA measurements in the same samples

  • Perform statistical analyses to determine significance of changes

This approach allows for reliable quantification of NCED9 across different experimental conditions.

What are the recommended fixation and immunohistochemistry protocols for localizing NCED9 in plant tissues?

Optimal immunohistochemistry protocol for NCED9 localization:

• Tissue preparation:

  • Fix fresh tissues in 4% paraformaldehyde in PBS (pH 7.4) for 12-16 hours at 4°C

  • For seeds: consider additional fixation time (24 hours) due to seed coat impermeability

  • Dehydrate tissues through graded ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Embed in paraffin or LR White resin for sectioning

• Sectioning and antigen retrieval:

  • Cut 5-8 μm sections for paraffin or 1-2 μm for resin

  • Deparaffinize and rehydrate sections if using paraffin

  • Perform antigen retrieval: 10mM sodium citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Cool slides slowly to room temperature

• Immunolabeling:

  • Block with 3% BSA, 0.3% Triton X-100 in PBS for 1 hour at room temperature

  • Incubate with primary NCED9 antibody (1:200-1:500 dilution) overnight at 4°C

  • Wash 3x with PBS + 0.1% Tween-20

  • Incubate with appropriate secondary antibody (1:500-1:1000)

  • Wash 3x with PBS + 0.1% Tween-20

  • Counterstain with DAPI (1:1000) for 5 minutes

  • Mount with anti-fade mounting medium

• Controls and validation:

  • Include no-primary-antibody control

  • Use nced9 mutant tissues as negative control

  • Compare with previously published expression patterns

  • Consider dual labeling with organelle markers (chloroplasts)

This protocol has been optimized based on general plant immunohistochemistry practices and the specific characteristics of NCED9 as a chloroplastic protein.

How do I resolve inconsistent results between antibody-based NCED9 detection and gene expression data?

When facing discrepancies between protein and transcript levels:

• Verify antibody specificity and sensitivity:

  • Re-test antibody specificity against recombinant NCED9

  • Check for potential cross-reactivity with other NCED family members

  • Determine detection limits using dilution series of recombinant protein

• Consider post-transcriptional regulation:

  • mRNA stability and translation efficiency may vary between conditions

  • Protein turnover rates may differ from transcript turnover

  • Analyze microRNA profiles that might target NCED9 transcripts

• Technical troubleshooting:

  • Optimize protein extraction for different tissues (especially seeds)

  • Verify RNA quality and appropriate reference genes for qPCR

  • Consider time-course experiments to capture delayed protein production

• Experimental validation:

  • Perform polysome profiling to assess translation efficiency

  • Use proteasome inhibitors to assess protein degradation rates

  • Implement pulse-chase experiments to measure protein half-life

A common cause of discrepancy is the temporal delay between transcription and protein accumulation. NCED9 activity is highly regulated post-translationally, particularly during seed development and stress responses .

What are the best approaches to study NCED9 protein modifications and their impact on enzyme activity?

To investigate NCED9 post-translational modifications:

• Phosphorylation analysis:

  • Immunoprecipitate NCED9 using specific antibodies

  • Perform western blotting with phospho-specific antibodies

  • Use mass spectrometry to identify phosphorylation sites

  • Compare phosphorylation patterns under different conditions

• Redox modification studies:

  • Use reducing/non-reducing SDS-PAGE to detect disulfide bonds

  • Apply alkylating agents to trap reduced/oxidized states

  • Investigate the impact of redox conditions on enzyme activity

  • Correlate with stress conditions known to alter cellular redox status

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation using NCED9 antibodies

  • Use yeast two-hybrid or split-GFP for interaction verification

  • Investigate interactions with other ABA biosynthesis enzymes

  • Study interaction with regulatory proteins under different conditions

• Enzyme activity assays:

  • Develop in vitro assays using immunopurified NCED9

  • Measure conversion of 9-cis-epoxycarotenoids to xanthoxin

  • Correlate enzyme activity with observed modifications

  • Compare wild-type and site-directed mutants of modification sites

These approaches help elucidate the complex regulation of NCED9 activity beyond transcriptional control, which is particularly important for understanding stress responses and developmental transitions in plants.

How should I interpret differences in NCED9 antibody staining patterns between wild-type and mutant plant tissues?

Interpreting immunostaining differences requires systematic analysis:

• Pattern analysis framework:

  • Document the cellular and subcellular localization patterns

  • Quantify signal intensity using appropriate image analysis software

  • Compare patterns across different developmental stages

  • Analyze co-localization with organelle markers

• Comparative assessment:

  • Compare with known expression patterns from promoter-reporter fusions

  • Correlate with in situ hybridization data for NCED9 mRNA

  • Contrast with other NCED family members' expression patterns

  • Reference against published data on NCED9 expression

• Mutant analysis considerations:

  • For nced9 null mutants: verify antibody specificity

  • For partial mutants: quantify reduced signal intensity

  • For regulatory mutants: assess altered tissue/cellular distribution

  • For stress/hormone treatments: document redistribution patterns

• Functional correlation:

  • Correlate staining patterns with ABA measurements

  • Link to phenotypic consequences (e.g., dormancy changes)

  • Connect to downstream ABA-responsive gene expression

  • Integrate with other ABA biosynthesis enzyme localizations

When analyzing seed tissues specifically, remember that NCED9 expression has been documented in both the embryo and endosperm at mid-development stages, with specific patterns in epidermal cells of the embryo but not in the endosperm at later stages .

What approaches can resolve conflicting data between different NCED9 antibodies used in the same experiment?

When different antibodies targeting the same protein yield inconsistent results:

• Technical validation:

  • Compare antibody specifications (polyclonal vs. monoclonal)

  • Verify epitope locations (N-terminal, C-terminal, internal)

  • Assess production methods (peptide vs. recombinant protein immunization)

  • Determine cross-reactivity profiles with other NCED family members

• Systematic comparison:

  • Perform side-by-side western blots under identical conditions

  • Use nced9 mutants as negative controls

  • Test with recombinant NCED9 protein as positive control

  • Compare detection sensitivities using dilution series

• Resolution strategies:

  • Consider protein conformation effects on epitope accessibility

  • Test alternative fixation/extraction protocols that may affect epitope exposure

  • Evaluate antibody performance under native vs. denaturing conditions

  • Assess potential post-translational modifications that might mask epitopes

• Validation through alternative approaches:

  • Use epitope-tagged NCED9 constructs (FLAG, HA, etc.)

  • Implement fluorescent protein fusions for localization studies

  • Combine with mass spectrometry for protein identification

  • Cross-validate with specific inhibitors of NCED activity

The conflicting data may reveal important biological insights about protein conformation, processing, or interactions rather than representing technical artifacts.

How can I distinguish between NCED9-specific signals and potential cross-reactivity with other NCED family members in complex plant samples?

Distinguishing specific NCED9 signals from cross-reactivity:

• Complementary control experiments:

  • Use tissues from nced9 single mutants to identify remaining cross-reactive signals

  • Test antibodies against multiple nced mutants (nced3, nced5, nced6, nced9)

  • Compare signal patterns with known expression profiles of each NCED gene

  • Analyze samples from plants overexpressing individual NCED genes

• Advanced analytical approaches:

  • Implement immunodepletion with recombinant NCED proteins

  • Perform sequential immunoprecipitation with different NCED antibodies

  • Use peptide competition assays with specific immunogenic peptides

  • Apply super-resolution microscopy to detect subtle localization differences

• Molecular size differentiation:

  • Develop high-resolution western blotting to separate similar-sized NCED proteins

  • Use 2D gel electrophoresis to separate NCEDs by both size and isoelectric point

  • Apply gradient gels for better separation of similarly sized proteins

  • Consider native gel electrophoresis to separate by conformation

• Computational analysis:

  • Create antibody specificity matrices based on epitope sequence conservation

  • Use prediction algorithms to identify potential cross-reactive regions

  • Implement image analysis algorithms to distinguish subtle pattern differences

  • Perform correlation analyses between signals and known expression patterns

The table below summarizes the expression patterns of different NCED family members to aid in distinguishing cross-reactivity:

How can NCED9 antibodies be used to investigate the relationship between ABA biosynthesis and other hormone signaling pathways?

Methodological approaches for hormone crosstalk studies:

• Co-immunoprecipitation studies:

  • Use NCED9 antibodies to pull down protein complexes

  • Identify interactions with components of other hormone pathways

  • Investigate how these interactions change during development or stress

  • Correlate with hormone measurements under the same conditions

• Dual immunolocalization:

  • Perform co-localization studies with antibodies against:

    • Gibberellin biosynthesis enzymes (given known ABA-GA antagonism in seeds)

    • Auxin transport or signaling components

    • Ethylene biosynthesis enzymes

  • Analyze spatial relationships between different hormone pathways

• Chromatin immunoprecipitation (ChIP) applications:

  • Use antibodies against transcription factors known to regulate NCED9

  • Investigate how hormone treatments affect TF binding to the NCED9 promoter

  • Analyze epigenetic modifications at the NCED9 locus after hormone treatments

  • Correlate with changes in NCED9 protein levels

• Hormone response time-course experiments:

  • Apply exogenous hormones and monitor NCED9 protein levels over time

  • Compare protein changes with transcriptional responses

  • Correlate with physiological effects (stomatal closure, growth inhibition)

  • Analyze in different genetic backgrounds (hormone signaling mutants)

This approach helps elucidate how NCED9-mediated ABA biosynthesis integrates with gibberellin signaling during seed development and dormancy regulation .

What insights can immunoprecipitation with NCED9 antibodies provide about protein-protein interactions in ABA biosynthesis?

Immunoprecipitation-based investigation of NCED9 interactions:

• Optimized IP protocol:

  • Harvest plant tissues at developmental stages with high NCED9 expression

  • Use mild extraction buffers to preserve protein-protein interactions

  • Cross-link proteins in vivo before extraction (optional)

  • Perform IP with NCED9 antibodies coupled to magnetic beads

  • Analyze by mass spectrometry for interacting partners

• Targeted interaction analysis:

  • Probe immunoprecipitates for other ABA biosynthesis enzymes

  • Investigate potential protein complexes with carotenoid biosynthesis enzymes

  • Test for interactions with regulatory proteins (kinases, phosphatases)

  • Analyze interactions with chloroplast proteins (NCED9 is chloroplastic)

• Dynamic interaction profiling:

  • Compare interactomes under normal vs. stress conditions

  • Analyze developmental stage-specific interactions (especially in seeds)

  • Investigate hormone treatment effects on protein-protein interactions

  • Assess how post-translational modifications affect interaction patterns

• Functional validation of interactions:

  • Confirm key interactions using yeast two-hybrid or BiFC assays

  • Test interaction mutants for effects on ABA biosynthesis

  • Analyze co-expression patterns of interacting proteins

  • Investigate subcellular co-localization of interaction partners

This approach can reveal novel regulatory mechanisms controlling NCED activity and ABA biosynthesis during development and stress responses.

How can advanced imaging techniques combined with NCED9 antibodies enhance our understanding of ABA biosynthesis regulation?

Cutting-edge imaging approaches for NCED9 research:

• Super-resolution microscopy applications:

  • Implement STORM or PALM imaging for nanoscale localization

  • Investigate NCED9 distribution within chloroplasts

  • Analyze co-localization with other ABA biosynthesis enzymes

  • Study potential microdomains of ABA production within organelles

• Live-cell imaging strategies:

  • Use cell-permeable labeled antibody fragments (Fab or nanobodies)

  • Combine with fluorescent ABA reporters for simultaneous visualization

  • Track dynamic changes in NCED9 localization during stress responses

  • Monitor protein turnover using photoactivatable fusion proteins

• Multi-modal imaging approaches:

  • Correlative light and electron microscopy (CLEM) to link function and ultrastructure

  • Combine with metabolite imaging (Raman microscopy) to correlate with ABA production

  • Implement FRET-FLIM to measure protein-protein interactions in vivo

  • Use optogenetics to manipulate ABA production while imaging responses

• Quantitative image analysis:

  • Apply machine learning algorithms for pattern recognition

  • Develop computational models of subcellular NCED9 distribution

  • Implement 3D reconstructions of NCED9 distribution in tissues

  • Correlate spatial patterns with transcriptional and physiological responses

These advanced approaches can reveal the spatial and temporal dynamics of NCED9 during ABA biosynthesis, particularly the coordination between different cellular compartments and the relationship with other signaling pathways.

What are the emerging applications of NCED9 antibodies in studying plant responses to climate change?

Novel applications for climate change research:

• Stress adaptation studies:

  • Use NCED9 antibodies to quantify protein levels under extreme temperature conditions

  • Compare NCED9 distribution patterns in drought-tolerant vs. sensitive varieties

  • Investigate the relationship between NCED9 levels and heat/drought recovery

  • Study epigenetic modifications at the NCED9 locus after stress memory formation

• Climate simulation experiments:

  • Monitor NCED9 protein dynamics under fluctuating stress conditions

  • Analyze protein-level adaptation to repeated stress cycles

  • Investigate the correlation between NCED9 protein levels and physiological adaptations

  • Compare ancestral vs. modern crop varieties for NCED9 expression patterns

• Multi-stress interaction analysis:

  • Study combined effects of drought, heat, and elevated CO₂ on NCED9 levels

  • Investigate how pathogen infection affects stress-induced NCED9 expression

  • Analyze NCED9 distribution during combined biotic and abiotic stress

  • Develop predictive models correlating NCED9 levels with stress tolerance

• Crop improvement applications:

  • Identify optimal NCED9 expression patterns for climate resilience

  • Screen germplasm collections for beneficial NCED9 variants

  • Analyze transgenic lines with modified NCED9 expression under field conditions

  • Investigate seed dormancy/germination adaptations to changing climate conditions

These approaches leverage NCED9 antibodies to understand the molecular basis of climate adaptation in plants, particularly through ABA-mediated responses to environmental stresses.

How might single-cell approaches utilizing NCED9 antibodies transform our understanding of ABA biosynthesis heterogeneity within tissues?

Single-cell analysis strategies:

• Single-cell immunocytochemistry:

  • Develop microdissection protocols for seed tissues

  • Apply NCED9 antibodies to individual isolated cells

  • Quantify cell-to-cell variability in protein expression

  • Correlate with developmental stage and cell identity

• Flow cytometry applications:

  • Optimize protoplast isolation from tissues of interest

  • Develop intracellular staining protocols for NCED9

  • Implement fluorescence-activated cell sorting (FACS)

  • Analyze sorted populations for NCED9 levels and post-translational modifications

• In situ proximity ligation assays:

  • Apply proximity ligation to detect NCED9 interactions at single-cell resolution

  • Investigate cell-specific interaction networks

  • Analyze spatial gradients of protein interactions across tissues

  • Correlate with ABA gradient formation during seed development

• Integration with single-cell transcriptomics:

  • Combine protein analysis with single-cell RNA-seq

  • Analyze correlation between mRNA and protein at single-cell level

  • Identify cell populations with unique NCED9 regulation patterns

  • Develop cellular resolution maps of ABA biosynthesis capacity

What potential exists for developing multiplexed antibody approaches to simultaneously track multiple NCED family members in plant tissues?

Multiplexed detection strategies:

• Multicolor immunofluorescence optimization:

  • Develop antibodies from different host species (rabbit, mouse, goat)

  • Select fluorophores with minimal spectral overlap

  • Implement sequential staining protocols when necessary

  • Apply spectral unmixing for closely overlapping signals

• Mass cytometry (CyTOF) adaptation:

  • Label NCED antibodies with different metal isotopes

  • Develop protocols for plant tissue preparation

  • Analyze multiple NCEDs simultaneously at single-cell resolution

  • Correlate with markers for cell identity and stress status

• Multiplex western blotting techniques:

  • Implement fluorescent western blotting with multiple channels

  • Use antibodies that target different epitopes with distinct sizes

  • Apply strip-and-reprobe techniques optimized for plant proteins

  • Develop computational tools for signal deconvolution

• Advanced microscopy approaches:

  • Apply multiplexed CODEX imaging for highly parallel detection

  • Develop cyclic immunofluorescence protocols for plant tissues

  • Implement array tomography for 3D reconstruction

  • Combine with tissue clearing techniques for whole-organ imaging

These approaches would allow researchers to track the spatiotemporal dynamics of the entire NCED family, revealing their coordinated roles in ABA biosynthesis during development and stress responses, particularly the complementary functions of NCED3, NCED5, NCED6, and NCED9 in different tissues and conditions .