EXPA14 Antibody

What is EXPA14 and why are antibodies against it valuable for plant biology research?

EXPA14 belongs to the α-expansin family of cell wall-loosening proteins that facilitate plant cell expansion through pH-dependent modification of cell wall components. Antibodies targeting EXPA14 are valuable research tools because they enable precise localization studies to determine where and when this protein functions during developmental processes. Unlike fluorescent protein fusion approaches that have proven challenging for expansin visualization (as seen with GFP fusions), antibodies can detect native EXPA14 protein in fixed tissue samples without concerns about fluorophore sensitivity to low pH environments . This makes EXPA14 antibodies essential for understanding the spatial regulation of cell expansion in roots, particularly at the transition zone/elongation zone boundary where EXPA14 shows dominant expression patterns.

How does EXPA14 expression pattern differ from other expansin family members?

EXPA14 displays a highly tissue-specific expression pattern that distinguishes it from other expansin family members. While EXPA1 shows strongest expression in the columella and lateral root cap of both primary and lateral roots, EXPA14 is predominantly expressed at the boundary between the transition zone and elongation zone . Additionally, EXPA14 demonstrates a different cellular distribution pattern between primary and lateral roots - in lateral roots, EXPA14 localizes not only to the cortex (as in primary roots) but also extends to the epidermal cell layers . This contrasts with EXPA15, which shows a relatively uniform pattern in the root apical meristem epidermis and emerging lateral roots. These distinct expression patterns suggest non-redundant functions among expansin family members in regulating tissue-specific cell expansion during root development.

What sample preparation techniques are most effective for EXPA14 antibody immunolocalization studies?

For effective immunolocalization of EXPA14 in plant tissues, researchers should implement a fixation protocol that preserves both protein antigenicity and cell wall structure. Begin with freshly harvested tissue fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 1-2 hours at room temperature. Following fixation, perform cell wall permeabilization using a combination of pectin-degrading enzymes (such as pectolyase and cellulase) to improve antibody penetration while maintaining tissue integrity. Critical steps include:

• Careful optimization of fixation duration to prevent overfixation that might mask epitopes

• Extended blocking steps (2-3 hours) with high BSA concentrations (3-5%) to reduce non-specific binding

• Extended primary antibody incubation (overnight at 4°C) with gentle agitation to ensure even penetration

• Multiple extensive washing steps between reagent applications

This approach addresses the particular challenges of immunolocalizing cell wall proteins like EXPA14, which may have limited accessibility due to their embedment within the complex cell wall matrix.

How can EXPA14 antibodies be used to investigate hormone-regulated cell expansion mechanisms?

EXPA14 antibodies offer powerful tools for investigating hormone-regulated cell expansion mechanisms in plants, particularly when combined with hormone treatment experiments. Research indicates that expansins, including EXPA14, are regulated by hormones that control cell expansion . To leverage EXPA14 antibodies in this context:

• Treat plants with specific hormones (auxin, gibberellin, brassinosteroids) at various concentrations and durations

• Process tissues for immunolocalization using optimized EXPA14 antibody protocols

• Quantify changes in EXPA14 distribution patterns and signal intensity

• Correlate hormone-induced cellular expansion with EXPA14 redistribution or abundance

• Perform Western blot analysis to quantify total EXPA14 protein levels following hormone treatments

This approach reveals how hormonal signals translate into changes in cell wall properties through EXPA14 activity. The specific localization of EXPA14 at the transition zone/elongation zone boundary makes it particularly valuable for examining how cells transition from the meristematic to the rapid elongation stage in response to hormonal cues. Researchers should include appropriate controls, including hormone transport inhibitors, to distinguish direct from indirect effects on EXPA14 regulation.

What strategies can address the challenges of detecting low-abundance EXPA14 in specific cell types?

Detecting low-abundance EXPA14 in specific cell types requires advanced approaches to enhance sensitivity while maintaining specificity:

Enhanced Signal Amplification Methods:

• Implement tyramide signal amplification (TSA) with the EXPA14 antibody, which can increase detection sensitivity by 10-100 fold over conventional immunodetection

• Utilize quantum dot-conjugated secondary antibodies, which provide superior photostability and brightness compared to conventional fluorophores

• Apply proximity ligation assay (PLA) techniques when investigating protein-protein interactions involving EXPA14

Cell Type-Specific Approaches:

• Combine EXPA14 immunodetection with fluorescence-activated cell sorting (FACS) of protoplasts from tissue-specific fluorescent marker lines

• Implement laser capture microdissection to isolate specific cell types prior to protein extraction and Western blot analysis

• Use tissue-specific promoters driving cell type markers in combination with EXPA14 antibody staining

These approaches help overcome detection limits when studying cell type-specific functions of EXPA14, particularly important given its differential expression between cortex and epidermal cell layers in lateral roots versus primary roots .

How can EXPA14 antibodies be utilized in biomechanical studies of cell wall properties?

EXPA14 antibodies can be instrumental in biomechanical studies of cell wall properties through several sophisticated experimental approaches:

Correlation of EXPA14 Localization with Mechanical Properties:

• Combine atomic force microscopy (AFM) measurement of cell wall stiffness with EXPA14 immunolocalization on the same tissue sections

• Create spatial maps correlating EXPA14 abundance with measured elastic modulus values at different developmental zones

• Track temporal changes in both EXPA14 distribution and cell wall mechanics during rapid growth phases

Functional Analysis:

• Apply EXPA14 antibodies to neutralize protein function in situ, then measure resultant changes in cell wall extensibility using micro-indentation or creep tests

• Perform pressure probe measurements of cell turgor in tissues with varying EXPA14 expression levels, as determined by immunolocalization

• Combine cell wall particle tracking with EXPA14 immunolabeling to examine the relationship between protein localization and wall expansion vectors

This multidisciplinary approach connects the molecular function of EXPA14 in the cell wall loosening process with measurable physical properties of the wall, providing deeper insights into how expansin activity translates to altered biomechanical properties during plant development.

What are the critical factors for generating specific polyclonal antibodies against EXPA14?

Generating highly specific polyclonal antibodies against EXPA14 requires careful consideration of several critical factors:

Antigen Design:

• Identify unique, surface-exposed epitopes within EXPA14 that differ from other expansin family members

• Avoid the highly conserved GH45-like domain and CBM63 domain shared across expansins

• Select peptide regions from the N-terminal or C-terminal regions with sequence divergence

• Consider using multiple peptide antigens to increase specificity and detection probability

Production and Purification:

• Express recombinant EXPA14 in bacterial systems with appropriate tags for purification (His-tag or GST-tag)

• Subject the protein to extensive purification to remove contaminating bacterial proteins

• Verify protein integrity through mass spectrometry before immunization

• Implement a multi-step immunization protocol with appropriate adjuvants to maximize antibody titer

Validation Steps:

• Perform extensive cross-reactivity testing against related expansins (particularly EXPA1, EXPA10, and EXPA15)

• Validate using tissues with known EXPA14 expression patterns, comparing with mCherry fusion localization data

• Confirm specificity using expa14 mutant tissues as negative controls

• Pre-absorb antibodies with recombinant proteins of closely related expansins to improve specificity

This comprehensive approach ensures the generation of antibodies that can reliably distinguish EXPA14 from other expansin family members, which is crucial given the high sequence similarity and overlapping expression domains of these proteins in plant tissues.

What are the optimal protein extraction methods for detecting EXPA14 in Western blots?

Extracting and detecting EXPA14 for Western blot analysis presents unique challenges due to its cell wall localization and relatively low abundance. Optimal extraction methods include:

Extraction Protocol:

• Rapidly freeze tissue in liquid nitrogen and grind to a fine powder

• Extract using a buffer containing:

  • High salt concentration (0.5-1.0 M NaCl) to disrupt ionic interactions with cell wall components

  • 1-2% Triton X-100 or similar non-ionic detergent

  • Cell wall-degrading enzymes (cellulase, pectolyase) at low concentrations

  • Protease inhibitor cocktail to prevent degradation

  • 50-100 mM sodium acetate buffer at pH 5.5 (near the pH optimum for expansin activity)

• Incubate extraction mixture with gentle agitation at 4°C for 2-3 hours

• Centrifuge at high speed (20,000 × g) to separate soluble proteins

• Concentrate extracted proteins using TCA precipitation or similar method

Western Blot Optimization:

• Use 10-12% SDS-PAGE gels for optimal resolution of EXPA14 (approximately 26-28 kDa)

• Include extended blocking times (2 hours minimum) with 5% non-fat milk or BSA

• Implement overnight primary antibody incubation at 4°C with gentle agitation

• Consider using high-sensitivity chemiluminescent detection systems or near-infrared fluorescent detection

• Include appropriate positive controls (recombinant EXPA14) and negative controls (extracts from expa14 knockout plants)

This approach specifically addresses the challenges of extracting EXPA14 from its native cell wall environment and detecting it reliably in Western blot applications.

How can researchers effectively perform co-immunoprecipitation with EXPA14 antibodies to identify interaction partners?

Performing effective co-immunoprecipitation (co-IP) with EXPA14 antibodies requires specialized approaches due to EXPA14's cell wall localization:

Optimized Co-IP Protocol:

• Cross-link proteins in intact tissues using membrane-permeable crosslinkers like DSP (dithiobis(succinimidyl propionate)) or formaldehyde at optimized concentrations

• Extract proteins using a two-phase approach:

  • Initial extraction with high salt and chelating agents to release ionically-bound cell wall proteins

  • Secondary extraction with cell wall-degrading enzymes to release covalently-bound proteins

• Pre-clear lysates extensively with protein A/G beads to reduce non-specific binding

• Immobilize anti-EXPA14 antibodies on activated beads using covalent coupling chemistry

• Perform extended incubation (overnight at 4°C) with gentle rotation

• Implement stringent washing steps with increasing salt concentrations

• Elute using competitive peptide elution rather than denaturing conditions when possible

• Analyze eluates with high-sensitivity mass spectrometry systems

Validation Approaches:

• Perform reverse co-IP with antibodies against identified interaction partners

• Verify interactions using alternative methods like bimolecular fluorescence complementation (BiFC)

• Include appropriate controls:

  • IgG control from the same species as the EXPA14 antibody

  • Extracts from expa14 mutant plants

  • Competition assays with recombinant EXPA14 protein

This detailed approach addresses the specific challenges of identifying proteins that interact with EXPA14 in its native cell wall environment, where protein-protein interactions may be transient or dependent on specific cell wall pH conditions.

How should researchers quantify and statistically analyze EXPA14 immunolocalization signals?

Accurate quantification and statistical analysis of EXPA14 immunolocalization signals requires rigorous methodological approaches:

Image Acquisition Guidelines:

• Maintain identical imaging parameters (exposure time, gain, offset) across all samples

• Capture z-stacks to account for the three-dimensional distribution of signals

• Include internal reference standards in each imaging session

• Image multiple biological and technical replicates (minimum n=5 for each condition)

Quantification Approach:

• Perform maximum intensity projections or sum slices depending on the biological question

• Define regions of interest (ROIs) based on anatomical landmarks rather than signal intensity

• Measure signal parameters including:

  • Mean fluorescence intensity within defined cell layers or structures

  • Signal density (number of punctate signals per unit area)

  • Co-localization coefficients with cell wall markers

  • Distance of signals from plasma membrane or other cellular structures

Statistical Analysis Framework:

• Test data for normality using Shapiro-Wilk or similar tests

• Apply appropriate statistical tests:

  • ANOVA with post-hoc tests for multiple condition comparisons

  • Mixed-effects models when analyzing nested data (e.g., multiple cells within tissues)

  • Non-parametric alternatives (Kruskal-Wallis, Mann-Whitney) when data violate normality assumptions

• Implement spatial statistics methods for analyzing distribution patterns

• Report effect sizes alongside p-values

This structured approach ensures reproducible and statistically sound analysis of EXPA14 immunolocalization patterns, particularly important when comparing expression across different developmental zones or treatments.

How can researchers reconcile contradictory data between EXPA14 antibody localization and reporter gene fusion approaches?

Reconciling contradictory data between antibody localization and reporter gene fusion approaches for EXPA14 requires systematic investigation of several potential explanations:

Experimental Design for Resolving Contradictions:

• Compare native promoter-driven EXPA14:mCherry fusions with antibody detection in the same tissues:

  • Process tissue sections for both simultaneous visualization where possible

  • Analyze multiple developmental stages and environmental conditions

  • Quantify signal patterns using standardized image analysis methods

• Investigate protein stability and turnover rates:

  • Perform pulse-chase experiments with protein synthesis inhibitors

  • Compare half-lives of native EXPA14 (detected by antibody) and fusion proteins

  • Examine whether fusion proteins are subject to different degradation pathways

• Assess potential artifacts in both approaches:

  • Test whether the fluorescent protein fusion affects protein targeting or function

  • Evaluate if antibody epitopes may be masked in specific cellular contexts

  • Determine if fixation procedures alter protein localization patterns

Explanatory Framework for Contradictions:

By systematically addressing these potential explanations, researchers can develop a more complete understanding of EXPA14 localization and function, recognizing that contradictions often reveal important biological insights about protein regulation and function.

What approaches can resolve discrepancies between EXPA14 protein levels detected by Western blot versus immunohistochemistry?

Resolving discrepancies between EXPA14 protein levels detected by Western blot versus immunohistochemistry requires methodical investigation of both technical and biological factors:

Systematic Troubleshooting Approach:

• Evaluate antibody performance in each application:

  • Test antibody specificity using recombinant EXPA14 protein spiked into native extracts

  • Determine if antibody recognizes native versus denatured epitopes differently

  • Assess optimal antibody concentrations for each application independently

• Examine extraction efficiency variables:

  • Compare different extraction protocols specifically optimized for cell wall proteins

  • Implement sequential extraction methods to capture all EXPA14 populations

  • Quantify extraction efficiency using recombinant EXPA14 recovery tests

• Investigate biological explanations:

  • Determine if tissue-specific post-translational modifications affect epitope recognition

  • Assess if EXPA14 forms different protein complexes in different cell types

  • Examine if subcellular localization patterns influence extraction or detection efficiency

Reconciliation Strategies:

• Develop complementary approaches:

  • Combine Western blot with mass spectrometry quantification

  • Implement tissue-specific extraction methods for Western blot analysis

  • Use flow cytometry of protoplasts from specific tissues with EXPA14 antibodies

• Standardize quantification methods:

  • Establish calibration curves using recombinant EXPA14 protein

  • Normalize signals to appropriate loading controls or reference proteins

  • Implement digital PCR for absolute quantification of transcript levels as a reference point

• Validate with genetic approaches:

  • Compare protein levels in overexpression lines versus wild-type

  • Analyze gradient series of knockdown lines with varying EXPA14 expression levels

By systematically addressing these technical and biological variables, researchers can develop a more comprehensive understanding of EXPA14 protein expression patterns and resolve apparent discrepancies between different detection methods.

How can EXPA14 antibodies be implemented in research on cell wall mechanobiology?

EXPA14 antibodies offer powerful tools for advancing research on cell wall mechanobiology through several innovative approaches:

Integration with Mechanical Testing:

• Combine immunolocalization of EXPA14 with cellular force microscopy to correlate protein distribution with mechanical properties

• Implement microindentation or nano-indentation testing in regions with varying EXPA14 abundance

• Use optical tweezers or magnetic particle-based approaches to apply localized forces while monitoring EXPA14 redistribution with immunofluorescence

Mechanical Perturbation Studies:

• Apply controlled mechanical stresses to tissues, then analyze EXPA14 redistribution using immunolocalization

• Investigate how mechanical signals alter EXPA14 synthesis, transport, and secretion patterns

• Develop antibody-based biosensors that can detect conformational changes in EXPA14 under different mechanical conditions

Mechanosignaling Integration:

• Examine co-localization of EXPA14 with mechanosensitive ion channels or other mechanosensors

• Analyze how mechanical perturbations affect EXPA14 phosphorylation or other post-translational modifications

• Determine if EXPA14 activity functions as part of mechanosensitive feedback loops regulating wall properties

This frontier research direction would significantly advance our understanding of how plants perceive and respond to mechanical forces through regulated cell wall modifications, with EXPA14 potentially serving as a key mediator between mechanical signals and wall extensibility changes.

What methodological advances could improve EXPA14 antibody applications in single-cell level research?

Advancing EXPA14 antibody applications to achieve single-cell resolution requires integration of emerging technologies:

Enhanced Imaging Approaches:

• Implement expansion microscopy protocols compatible with EXPA14 antibodies to achieve super-resolution imaging of cell wall localization

• Adapt CLARITY or similar tissue clearing methods for plant tissues while preserving antibody epitopes

• Apply correlative light and electron microscopy (CLEM) to precisely localize EXPA14 at the ultrastructural level

• Utilize adaptive optics to improve imaging depth and resolution in thick plant tissue sections

Single-Cell Analytical Methods:

• Develop microfluidic approaches for single-cell protein extraction followed by ultrasensitive ELISA detection of EXPA14

• Implement single-cell mass cytometry (CyTOF) with metal-conjugated EXPA14 antibodies

• Adapt proximity ligation assays for single-molecule detection of EXPA14 in intact tissues

• Combine single-cell RNA sequencing with antibody detection (CITE-seq adaptation) to correlate transcription with protein levels

Live-Cell Applications:

• Develop cell-permeable nanobodies derived from EXPA14 antibodies for live-cell imaging

• Create split-fluorophore systems where one component is fused to an EXPA14-targeting antibody fragment

• Implement optogenetic approaches to manipulate EXPA14 function with spatiotemporal precision

These methodological advances would transform our ability to study EXPA14 dynamics at unprecedented resolution, enabling researchers to address fundamental questions about cell-to-cell variability in expansin function and localization during plant development.

How might EXPA14 antibodies contribute to understanding evolutionary adaptations in plant cell walls?

EXPA14 antibodies offer valuable tools for comparative evolutionary studies of plant cell wall adaptations across species:

Cross-Species Comparative Studies:

• Evaluate antibody cross-reactivity with EXPA14 homologs across diverse plant lineages

• Analyze conservation of expression patterns in equivalent tissues across phylogenetically diverse species

• Compare subcellular localization patterns to identify evolutionarily conserved versus divergent features

• Correlate EXPA14 localization with cell wall composition variations across species

Adaptation to Environmental Conditions:

• Examine how EXPA14 expression and localization patterns change in response to various stresses across species with different ecological adaptations

• Compare EXPA14 distribution in closely related species adapted to different environmental niches

• Analyze whether EXPA14 shows different pH sensitivities or cell wall binding properties across species from various habitats

Methodological Approach:

• Develop a panel of antibodies targeting conserved versus variable regions of EXPA14

• Implement standardized immunolocalization protocols applicable across diverse plant species

• Combine with glycome profiling to correlate EXPA14 localization with cell wall polysaccharide distribution patterns

• Integrate with comparative genomics and structural biology approaches

This evolutionary perspective would provide insights into how expansin-mediated cell wall modifications have evolved as adaptation strategies, potentially revealing fundamental principles about plant structural evolution and identifying key innovations in cell wall biology across plant lineages.