At4g16260 Antibody

What is At4g16260 and what cellular function does it serve?

At4g16260 encodes a Glycoside Hydrolase (GH) family 17 protein in Arabidopsis thaliana that is involved in plant cell wall metabolism. Based on proteomic analysis of elongating cells, this protein is developmentally regulated, appearing specifically in 11-day samples but not in 5-day samples . As a member of the GH family 17, it likely functions in the hydrolysis of glycosidic bonds in complex carbohydrates, potentially contributing to cell wall remodeling during plant growth and development. The protein's developmentally-specific expression pattern suggests it plays a specialized role during particular stages of cell elongation or maturation in Arabidopsis.

How does At4g16260 expression change during plant development?

Proteomic analysis demonstrates that At4g16260 exhibits a distinct developmental expression pattern. The protein is absent in 5-day samples but clearly detectable in 11-day samples of elongating cells . This temporal regulation suggests that At4g16260 functions during the later stages of cell wall development. Unlike some other cell wall proteins that show consistent expression across developmental stages (marked with "≈" in comparative analyses), At4g16260 shows a clear on/off pattern, indicating tight transcriptional or translational control mechanisms that regulate its expression during specific developmental windows. This distinct temporal regulation makes it a valuable marker for studying developmental transitions in cell wall composition.

How does At4g16260 relate to other cell wall-modifying proteins?

At4g16260 belongs to the GH family 17 of proteins that act on carbohydrates, but it functions within a complex network of diverse cell wall-modifying enzymes. Comparative analysis reveals that multiple protein families contribute to cell wall dynamics during plant development, including:

• Other GH family members (GH16/XTHs, GH28/polygalacturonases)

• Expansins (facilitate cell wall loosening)

• Peroxidases (involved in cross-linking)

• Proteases (multiple families showing developmental regulation)

• Various structural proteins

Within this network, At4g16260 shows a unique expression pattern compared to other GH family proteins like XTHs (xyloglucan endotransglucosylase/hydrolases), which exhibit more complex patterns of expression across developmental stages. This suggests that At4g16260 serves a specialized function during a specific developmental window rather than contributing to general cell wall maintenance.

What are the recommended protocols for immunolocalization of At4g16260 in plant tissues?

For effective immunolocalization of At4g16260 in plant tissues, researchers should implement the following protocol:

• Tissue fixation: Fix fresh Arabidopsis tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at room temperature or overnight at 4°C.

• Embedding and sectioning: After dehydration through an ethanol series, embed tissues in paraffin or resin. For paraffin, cut 7-10 μm sections; for resin, cut 1-2 μm sections.

• Antigen retrieval: This step is often critical for cell wall proteins. Treat sections with 0.1% pectolyase or 0.1M citrate buffer (pH 6.0) at 37°C for 10-20 minutes.

• Blocking: Incubate sections with 3% BSA in PBS for 1 hour at room temperature.

• Primary antibody application: Apply the polyclonal At4g16260 antibody at a dilution of 1:100 to 1:500 in blocking solution. Incubate overnight at 4°C in a humid chamber.

• Detection system: Use fluorescent-labeled secondary antibodies (1:200-1:500) for 1-2 hours at room temperature, or develop with an appropriate enzymatic detection system.

• Controls: Always include negative controls (omitting primary antibody) and consider using tissues from different developmental stages (5-day vs. 11-day) as biological controls based on the known expression pattern .

When analyzing results, pay particular attention to cell types undergoing active elongation or maturation, as the protein shows developmental specificity in its expression.

How can western blotting be optimized for detecting At4g16260 protein?

To optimize western blotting for At4g16260 detection, researchers should follow these methodological guidelines:

• Sample preparation: Extract cell wall proteins using a sequential extraction protocol:

  • Extract with 0.2M CaCl₂ (1:4 w/v) for 2 hours at 4°C

  • Follow with 2M LiCl extraction for ionically-bound proteins

  • Consider additional extraction with SDS buffer for more tightly bound proteins

• Protein quantification: Use Bradford or BCA assay with BSA standard curve, adjusting for the presence of extraction reagents.

• Gel electrophoresis: Run 10-20 μg protein per lane on 10-12% SDS-PAGE gels. Cell wall proteins often show anomalous migration due to glycosylation, so include molecular weight markers.

• Membrane transfer: Use PVDF membranes for glycoproteins, transferring at 100V for 1 hour or 30V overnight at 4°C.

• Blocking: Block with 5% non-fat milk in TBST for 1 hour, or consider 3% BSA if milk proteins interfere.

• Antibody incubation: Apply polyclonal At4g16260 antibody at 1:1000 dilution overnight at 4°C.

• Developmental controls: Include samples from both 5-day and 11-day tissues as expression controls, expecting signal only in 11-day samples .

• Detection: Use ECL substrate with exposure times ranging from 30 seconds to 5 minutes, as cell wall proteins can vary in abundance.

When troubleshooting, remember that At4g16260 is developmentally regulated, so confirm your tissue source is at the appropriate developmental stage (11 days rather than 5 days for detection) .

What considerations are important when using the At4g16260 antibody for co-immunoprecipitation studies?

When performing co-immunoprecipitation (Co-IP) with the At4g16260 antibody, researchers should consider these methodological aspects:

• Buffer optimization: Cell wall proteins require specialized extraction conditions. Use a buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Nonidet P-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Crosslinking considerations: Since protein-protein interactions at the cell wall may be transient, consider using a crosslinking agent like formaldehyde (1% for 10 minutes) or DSP (dithiobis(succinimidyl propionate)) before extraction.

• Pre-clearing: Pre-clear lysates with Protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

• Antibody binding: Incubate At4g16260 polyclonal antibody with Protein A/G beads first (2-5 μg antibody per reaction), then add to pre-cleared lysate.

• Washing stringency: Determine optimal washing stringency empirically; start with 4-5 washes using buffer containing 150 mM NaCl, then adjust salt concentration based on results.

• Elution method: Use either acidic elution (0.1 M glycine, pH 2.5) or SDS sample buffer heating, depending on downstream applications.

• Controls: Include:

  • IgG control from the same species as the At4g16260 antibody

  • Input sample (5-10% of starting material)

  • Lysate from 5-day tissues as a negative control (where the protein is not expressed)

• Verification: Confirm successful pull-down by western blotting a portion of the immunoprecipitate.

When analyzing potential interaction partners, focus on other proteins that show similar developmental expression patterns (11-day presence) and co-localize in cell walls, such as other GH family members or cell wall remodeling enzymes expressed at the same developmental stage .

How should researchers interpret At4g16260 expression data across different developmental stages?

When interpreting At4g16260 expression data across developmental stages, researchers should apply these analytical principles:

• Temporal pattern assessment: The definitive expression pattern of At4g16260 shows absence at 5 days and presence at 11 days of development . This binary pattern differs from other cell wall proteins showing more nuanced expression changes, suggesting:

  • Tight transcriptional regulation at a specific developmental window

  • Potential role in a stage-specific cell wall modification event

  • Possible involvement in the transition from primary to secondary wall formation

• Developmental context: Interpret expression within the broader context of cell wall protein dynamics. Compare with expression patterns of:

  • Other GH family members (some show opposite patterns, being present only at 5 days)

  • Peroxidases (showing diverse developmental patterns)

  • Structural proteins (many show consistent expression across stages)

• Quantitative analysis framework: When using proteomics or transcriptomics data:

  • For absence/presence data: use statistical methods that handle zero values appropriately

  • For quantitative differences: normalize to internal standards consistently across experiments

  • Consider fold-change thresholds in combination with statistical significance

• Integration with physiological data: Correlate expression profiles with cell elongation rates, wall mechanical properties, or metabolomic profiles of cell wall components to establish functional relationships.

The developmental restriction of At4g16260 to 11-day samples provides a valuable internal control for experimental validation . Researchers can use this well-established pattern to verify their experimental systems before conducting more complex analyses.

What are the best approaches for analyzing potential functional redundancy between At4g16260 and other glycoside hydrolases?

To analyze potential functional redundancy between At4g16260 and other glycoside hydrolases, researchers should implement these methodological approaches:

• Phylogenetic analysis:

  • Construct phylogenetic trees of the complete GH family 17 in Arabidopsis

  • Identify the closest paralogs to At4g16260

  • Analyze conserved domains and active sites across family members

• Expression correlation analysis:

  • Compare expression patterns across developmental stages and tissues

  • Look for GH17 members with complementary expression patterns to At4g16260

  • Analyze the family-wide expression table to identify potential functional groupings

• Substrate specificity assessment:

  • Compare predicted substrate binding pockets across family members

  • When available, use enzymatic assay data to group functionally similar enzymes

  • Consider the distinct developmental presence of At4g16260 at 11 days compared to other GH members

• Genetic approach:

  • Design experiments using single, double, and higher-order mutants of related GH17 family members

  • Phenotype these mutants for cell wall defects, particularly at 11 days of development

  • Apply statistical methods that can detect genetic interactions (synergistic vs. additive effects)

• Network analysis:

  • Build co-expression networks including At4g16260 and other cell wall proteins

  • Use weighted gene co-expression network analysis (WGCNA) to identify modules of functionally related genes

  • Compare the network position of At4g16260 with other GH family members

When interpreting results, consider that At4g16260's specific developmental expression profile (present at 11 days, absent at 5 days) may indicate specialized, non-redundant functions despite similarity to other GH family members. Its distinct temporal regulation suggests unique functions even among closely related enzymes.

How do you interpret contradictory results between antibody-based detection and transcriptomic data for At4g16260?

When facing contradictory results between antibody-based detection and transcriptomic data for At4g16260, researchers should apply this analytical framework:

• Temporal dynamics consideration:

  • Transcript levels often precede protein accumulation by hours or days

  • For cell wall proteins, post-translational modification and trafficking can delay detection

  • Compare sampling timepoints carefully - the sharp developmental transition (absent at 5 days, present at 11 days) might appear differently at the transcript level

• Methodological assessment:

  • Antibody specificity: Verify the At4g16260 antibody doesn't cross-react with related GH family members

  • RNA preparation: Ensure RNA extraction methods adequately capture transcripts from all cell types

  • Normalization methods: Different normalization approaches in transcriptomics can affect apparent expression levels

• Biological explanations to consider:

  • Post-transcriptional regulation: miRNAs might target At4g16260 transcripts

  • Protein stability: The protein might accumulate despite lower transcript levels due to high stability

  • Tissue-specific expression: Bulk tissue analysis might mask cell-specific expression patterns

• Validation approach:

  • Perform qRT-PCR with multiple reference genes for transcript quantification

  • Use multiple antibody-based methods (western blot, immunolocalization, ELISA)

  • Include appropriate developmental stage controls (5-day vs. 11-day samples)

  • Consider in situ hybridization to localize transcripts in specific cell types

• Integrated data analysis:

  • Develop a mathematical model that accounts for transcription rates, translation efficiency, and protein turnover

  • Apply statistical methods specifically designed for integrating multi-omics data

  • Consider Bayesian approaches to weight evidence from different experimental methods

Remember that the proteomics data clearly shows At4g16260 protein is detectable at 11 days but not at 5 days of development , providing a robust reference point for resolving contradictions with transcriptomic data.

What are common technical challenges when working with the At4g16260 antibody and how can they be resolved?

Researchers working with the At4g16260 antibody may encounter these technical challenges, which can be resolved through methodological adjustments:

• High background signal in immunolocalization:

  • Cause: Insufficient blocking or non-specific binding

  • Resolution: Increase blocking time to 2 hours; try different blocking agents (5% BSA, 5% normal serum, commercial blocking reagents); add 0.1% Tween-20 to washing steps; optimize antibody dilution (try 1:500 to 1:2000 range)

• Weak or absent signal in western blots:

  • Cause: Insufficient protein extraction, inappropriate developmental stage, or protein degradation

  • Resolution: Verify tissue source is from 11-day (not 5-day) samples ; use specialized cell wall protein extraction buffers; include protease inhibitors; increase antibody concentration or incubation time; try enhanced chemiluminescence detection systems

• Multiple bands in western blots:

  • Cause: Glycosylation variants, proteolytic processing, or cross-reactivity

  • Resolution: Pre-treat samples with deglycosylation enzymes; verify with knockout/knockdown controls; perform peptide competition assays; consider using multiple antibodies targeting different epitopes

• Inconsistent immunoprecipitation results:

  • Cause: Buffer incompatibility, weak antibody-antigen interaction, or co-factor requirements

  • Resolution: Test different IP buffers; crosslink antibody to beads; try native vs. denaturing conditions; verify antibody batch consistency; include appropriate controls (IgG, 5-day samples)

• Discrepancies between labs or experiments:

  • Cause: Growth condition variations, antibody lot differences, or protocol variations

  • Resolution: Standardize growth conditions; validate with molecular markers of developmental stages; create detailed SOPs; use consistent antibody dilutions; incorporate positive and negative controls in every experiment

When troubleshooting, remember that At4g16260 shows a distinct developmental expression pattern (absent at 5 days, present at 11 days) , which provides a valuable internal control for validating experimental conditions. Always include samples from both developmental stages as biological controls.

How can researchers effectively extract and preserve cell wall proteins like At4g16260 for antibody-based studies?

For effective extraction and preservation of cell wall proteins like At4g16260, researchers should implement this comprehensive methodology:

• Sequential extraction protocol:

  • Step 1: Grind tissue in liquid nitrogen to fine powder

  • Step 2: Extract with 0.2M CaCl₂ buffer (100 mM Tris-HCl pH 8.0, 0.2M CaCl₂, 5 mM DTT) at 4°C for 2 hours with gentle agitation

  • Step 3: Centrifuge (10,000g, 15 min) and collect supernatant

  • Step 4: Re-extract pellet with 2M LiCl buffer (100 mM Tris-HCl pH 8.0, 2M LiCl, 5 mM DTT)

  • Step 5: For tightly bound proteins, extract remaining pellet with SDS buffer (100 mM Tris-HCl pH 8.0, 2% SDS, 10 mM DTT)

• Protein preservation strategies:

  • Add protease inhibitor cocktail to all extraction buffers

  • Maintain samples at 4°C throughout extraction

  • Add 10% glycerol to final protein samples for freezing stability

  • Aliquot samples to avoid freeze-thaw cycles

  • For long-term storage, flash-freeze in liquid nitrogen and store at -80°C

• Sample concentration methods:

  • Use Amicon Ultra centrifugal filters (10 kDa MWCO) for concentration

  • Alternatively, use TCA/acetone precipitation (add 4 volumes of 10% TCA in acetone, incubate at -20°C overnight)

  • For small samples, consider carrier-assisted precipitation (add 1 μg/μL glycogen)

• Quality control checkpoints:

  • Verify protein integrity via silver-stained SDS-PAGE before antibody applications

  • Include known cell wall markers as extraction efficiency controls

  • Perform Bradford or BCA assays for protein quantification

  • Verify sample pH is appropriate (pH 6.8-8.0) for downstream applications

• Developmental considerations:

  • Precisely stage plant materials (5-day vs 11-day samples show dramatic differences)

  • Document growth conditions that may affect cell wall composition

  • Consider tissue-specific extraction for organs with different cell wall compositions

This methodology accounts for the specialized nature of cell wall proteins and their developmental regulation, particularly important for At4g16260 which shows clear developmental specificity (present at 11 days, absent at 5 days) .

What controls are essential when validating antibody specificity for At4g16260 protein?

When validating antibody specificity for At4g16260 protein, researchers must implement these essential controls:

• Genetic controls:

  • Knockout/knockdown lines: Test antibody against At4g16260 T-DNA insertion lines or CRISPR-Cas9 generated knockouts (signal should be absent)

  • Overexpression lines: Test against plants overexpressing At4g16260 (signal should be enhanced)

  • Wild-type samples: Include both Col-0 and other ecotypes to assess natural variation

• Developmental stage controls:

  • Negative tissue control: Use 5-day samples where the protein is known to be absent

  • Positive tissue control: Use 11-day samples where the protein is known to be present

  • Developmental series: Test intermediate timepoints to establish expression dynamics

• Technical validation controls:

  • Peptide competition assay: Pre-incubate antibody with the immunizing peptide (should abolish signal)

  • Secondary antibody only: Omit primary antibody to assess background (no specific signal should be detected)

  • Cross-reactivity test: Test against recombinant proteins from closely related GH17 family members

• Methodology-specific controls:

  • For western blotting: Include molecular weight markers and verify expected size

  • For immunolocalization: Include known cell wall markers with established patterns

  • For IP experiments: Include IgG control from the same species as the antibody

• Antibody validation documentation:

  • Record antibody lot number, dilution, and incubation conditions

  • Document all optimization steps with appropriate positive and negative controls

  • Maintain a validation worksheet with quantitative signal-to-noise ratios across conditions

The developmental regulation of At4g16260 (absent at 5 days, present at 11 days) provides a particularly valuable internal control system that should be leveraged in all validation experiments. This natural on/off switch in expression helps establish specificity in a biologically relevant context.

How can researchers use the At4g16260 antibody to investigate cell wall remodeling during development?

To investigate cell wall remodeling during development using the At4g16260 antibody, researchers should implement these advanced methodological approaches:

• High-resolution temporal analysis:

  • Sample tissues at 24-hour intervals between days 5-11 to pinpoint the exact timing of At4g16260 appearance

  • Correlate protein accumulation with specific developmental events and morphological changes

  • Combine with time-course transcriptomics to establish the full regulatory cascade

• Spatial mapping of protein distribution:

  • Perform confocal immunofluorescence to map At4g16260 distribution across tissue types

  • Use tissue-specific markers to correlate expression with tissue identity

  • Apply super-resolution microscopy techniques (STED, STORM) to determine nanoscale localization within cell wall layers

  • Compare with the distribution patterns of other developmentally regulated cell wall proteins

• Co-localization with cell wall components:

  • Perform dual-labeling with At4g16260 antibody and fluorescently tagged cell wall components

  • Use carbohydrate-binding modules (CBMs) to label specific polysaccharides

  • Analyze Pearson's correlation coefficients and Manders' overlap coefficients quantitatively

  • Correlate At4g16260 localization with wall mechanical properties using atomic force microscopy

• Environmental response profiling:

  • Subject plants to abiotic stresses (drought, salt, heat) and analyze changes in At4g16260 expression pattern

  • Test effects of phytohormones known to influence cell expansion (auxin, brassinosteroids, gibberellins)

  • Create a matrix of environmental conditions × developmental stages to establish regulatory networks

• Integrative multi-omics approach:

  • Combine proteomics, glycomics, and immunolocalization data in the same samples

  • Apply multivariate statistical methods to establish correlations between At4g16260 levels and specific wall components

  • Develop predictive models of cell wall assembly incorporating the developmental switch in At4g16260 expression

This comprehensive approach leverages the unique developmental regulation of At4g16260 (absent at 5 days, present at 11 days) as a precise marker for studying the transition in cell wall properties during plant development.

What advanced experimental approaches can resolve contradictions between functionally redundant cell wall proteins?

To resolve contradictions between functionally redundant cell wall proteins like At4g16260 and related glycoside hydrolases, researchers should implement these advanced experimental approaches:

• CRISPR-Cas9 multiplexing strategy:

  • Generate combinatorial knockout lines targeting At4g16260 and related GH17 family members

  • Apply statistical interaction modeling to phenotypic data to distinguish between:

    • Additive effects (suggesting independent functions)

    • Synergistic effects (suggesting cooperative functions)

    • Epistatic relationships (suggesting hierarchical functions)

  • Focus analysis on the 11-day developmental stage when At4g16260 is naturally expressed

• Domain-swapping chimeric proteins:

  • Design chimeric constructs swapping catalytic and binding domains between At4g16260 and related GH proteins

  • Express under native promoters in respective mutant backgrounds

  • Quantify complementation efficiency to map functional domains

  • Correlate with the developmental expression patterns documented in proteomic studies

• Substrate specificity profiling:

  • Express and purify recombinant At4g16260 and related GH proteins

  • Perform systematic enzyme kinetics with a diverse glycan microarray

  • Apply Michaelis-Menten analysis to establish substrate preferences

  • Correlate kinetic parameters with in vivo expression patterns at different developmental stages

• In situ activity labeling:

  • Develop activity-based protein profiling (ABPP) probes for GH enzymes

  • Apply to tissues at 5-day and 11-day stages

  • Compare activity patterns with immunolocalization patterns

  • Correlate with cell wall compositional changes using glycome profiling

• Systems biology modeling:

  • Construct a mathematical model incorporating enzyme-substrate relationships

  • Parameterize the model with experimental kinetic data

  • Simulate the effects of removing individual or multiple enzymes

  • Validate predictions with targeted experimental manipulations

This integrated approach combines genetic, biochemical, and computational methods to disentangle the complex functional relationships among cell wall proteins with potentially overlapping functions, using the developmental regulation of At4g16260 as a key reference point .

How can researchers leverage At4g16260 antibody for studying protein-carbohydrate interactions in the plant cell wall?

For studying protein-carbohydrate interactions in the plant cell wall using the At4g16260 antibody, researchers should implement these advanced methodological approaches:

• In situ proximity ligation assay (PLA):

  • Combine At4g16260 antibody with carbohydrate-binding modules (CBMs) targeting specific glycans

  • Apply PLA protocol to visualize close associations (<40 nm) in fixed tissue sections

  • Quantify signal intensity across different developmental stages and cell types

  • Compare interaction patterns at 5-day versus 11-day stages, expecting signals only in the latter

• Co-immunoprecipitation with glycan analysis:

  • Perform IP with At4g16260 antibody under native conditions

  • Analyze co-precipitated carbohydrates using mass spectrometry

  • Compare glycan profiles between developmental stages

  • Correlate findings with the developmental expression pattern of At4g16260

• Förster resonance energy transfer (FRET) microscopy:

  • Label At4g16260 antibody with donor fluorophore

  • Label carbohydrate-binding probes with acceptor fluorophore

  • Measure FRET efficiency to determine binding dynamics in vivo

  • Calculate spatial and temporal parameters of protein-carbohydrate interactions

• Surface plasmon resonance (SPR) analysis:

  • Immobilize purified At4g16260 protein on SPR chips

  • Flow defined oligosaccharides over the surface

  • Determine binding constants and kinetic parameters

  • Compare with other GH17 family members to establish specificity

• Cryo-electron microscopy:

  • Use immunogold labeling with At4g16260 antibody on high-pressure frozen samples

  • Perform tomographic reconstruction to map 3D distribution in the cell wall

  • Correlate with known cell wall architectures at different developmental stages

  • Compare with distribution patterns of other cell wall proteins from the same developmental stage

This comprehensive approach leverages the unique developmental expression pattern of At4g16260 (absent at 5-day, present at 11-day samples) to precisely study the dynamics of protein-carbohydrate interactions during critical developmental transitions in plant cell walls.

What are the most significant recent advances in understanding the function of At4g16260 in plant development?

The most significant recent advances in understanding At4g16260 function come from integrative approaches combining proteomics, genetics, and cell biology. Proteomic studies have established that At4g16260, a GH family 17 protein, shows a distinct developmental regulation pattern, being absent in 5-day samples but clearly detected in 11-day samples of elongating cells . This precise developmental timing suggests it plays a specialized role in cell wall remodeling during the transition from primary growth to a more mature cell wall state.

Furthermore, comparative proteomics has revealed that At4g16260 functions within a complex network of cell wall proteins with diverse developmental regulation patterns. While some proteins show consistent expression across developmental stages, others exhibit stage-specific expression, creating a dynamic protein landscape that orchestrates cell wall remodeling . These findings highlight the importance of studying At4g16260 not in isolation but as part of a coordinated developmental program involving multiple enzyme families.

The availability of specific antibodies against At4g16260 has enabled more detailed studies of its localization and dynamics, facilitating a deeper understanding of its function in specific cell types and developmental contexts. Looking forward, the integration of these approaches with advanced imaging, genetic manipulation, and biochemical characterization promises to further clarify the precise role of At4g16260 in plant cell wall development.

What future research directions should be prioritized regarding At4g16260 antibody applications?

Future research regarding At4g16260 antibody applications should prioritize these key directions:

• Single-cell proteomics applications: Develop protocols using the At4g16260 antibody for cell-specific isolation and analysis, enabling the study of cell wall protein dynamics at the single-cell level rather than in bulk tissues. This would provide unprecedented resolution of the developmental regulation observed in current studies .

• Live-cell imaging adaptations: Develop non-invasive approaches using modified antibody fragments (Fab, nanobodies) that can function in living cells, enabling real-time tracking of At4g16260 dynamics during development and in response to environmental stimuli.

• Antibody-enabled protein complex characterization: Apply advanced immunoprecipitation combined with mass spectrometry to identify the complete interactome of At4g16260, focusing on the 11-day developmental stage when the protein is expressed . This would reveal functional protein networks regulating cell wall assembly.

• Cross-species comparative immunology: Validate the At4g16260 antibody across related plant species to establish evolutionary conservation of expression patterns and enable comparative studies of cell wall development across diverse plant lineages.

• Therapeutic and agricultural applications: Explore the potential of At4g16260 antibodies for modifying plant growth characteristics through targeted immunomodulation, potentially creating new approaches for crop improvement by manipulating cell wall properties.