EXPA17 Antibody

What is EXPA17 and why is it significant in plant research?

EXPA17 is a non-hydrolytic cell wall-loosening protein belonging to the expansin family in Arabidopsis thaliana. It plays a crucial role in lateral root (LR) formation by facilitating cell separation that enables lateral root emergence through overlaying tissues of the primary root. EXPA17 is regulated by the transcription factor LBD18/ASL20, which directly increases EXPA17 transcript levels to promote lateral root development . The significance of EXPA17 lies in its role as a molecular mediator of hormone-induced developmental processes, particularly in the auxin signaling pathway that controls root architecture development. Understanding EXPA17 function provides insights into fundamental mechanisms of plant growth regulation and environmental adaptation through root system modification.

What methods are available for detecting EXPA17 protein expression in plant tissues?

To detect EXPA17 protein expression in plant tissues, researchers can employ several complementary approaches:

• Immunolocalization with specific antibodies: Using EXPA17-specific antibodies for immunohistochemistry or immunofluorescence microscopy to visualize protein distribution in tissue sections.

• Fluorescent protein fusion constructs: Creating EXPA17:GFP or EXPA17:mCherry fusion proteins expressed under native promoters (pEXPA17::EXPA17:GFP), similar to the approach used for EXPA1 and EXPA15 visualization .

• Promoter-reporter gene systems: Although these don't detect the protein directly, pEXPA17::GUS constructs can reveal the tissue-specific activity of the EXPA17 promoter, as demonstrated in studies where β-Glucuronidase (GUS) expression under the EXPA17 promoter was detected only in the roots of wild-type plants and was reduced in the LR primordium in an lbd18 mutant background .

• Western blot analysis: Using EXPA17-specific antibodies to detect and quantify protein levels in tissue extracts.

For optimal results, a combination of these approaches is recommended to validate findings across multiple detection methods.

What strategies can be used to develop specific antibodies against EXPA17?

Developing specific antibodies against EXPA17 requires careful antigen design and validation strategies:

• Antigen design outside conserved domains: To generate antibodies specific to EXPA17 rather than cross-reactive with other expansin family members, computational analysis should be used to identify unique epitopes outside of annotated domains that are conserved across expansins . This approach is crucial for avoiding cross-reactivity with highly similar expansin proteins.

• Recombinant protein expression: Express either full-length EXPA17 or selected unique peptide regions in E. coli expression systems with appropriate purification tags. This provides pure antigen for immunization or phage display selection .

• Synthetic antibody production via phage display: High-throughput pipeline approaches involving phage display can be particularly effective, as they allow for the screening of large antibody libraries against the target antigen under controlled conditions . This method has successfully generated antibodies against RNA-binding proteins and could be adapted for EXPA17.

• Verification of specificity: Any developed antibody must be tested against both wild-type and expa17 mutant or knockdown plants to confirm specificity. Additional testing against closely related expansins (EXPA1, EXPA10, EXPA14, EXPA15) should be performed to rule out cross-reactivity .

The development process should include rigorous testing for the antibody's performance in multiple applications (Western blotting, immunoprecipitation, immunofluorescence) to ensure versatility for research applications.

How can I validate the specificity of an EXPA17 antibody?

Validating EXPA17 antibody specificity requires a multi-faceted approach:

• Genetic validation: The most definitive validation comes from testing the antibody against tissues from wild-type plants versus expa17 knockout or knockdown lines. A specific antibody should show significantly reduced or absent signal in mutant plants .

• Recombinant protein controls: Testing the antibody against purified recombinant EXPA17 protein as a positive control, alongside other recombinant expansin family members to assess potential cross-reactivity.

• Pre-absorption tests: Pre-incubating the antibody with excess EXPA17 antigen should abolish specific signals in subsequent applications, confirming specificity.

• Western blot analysis: Confirming that the antibody detects a single band of the expected molecular weight (~25-30 kDa for EXPA17) in wild-type plant extracts, which is absent or reduced in expa17 mutants.

• Immunoprecipitation efficiency test: Verifying that the antibody can successfully immunoprecipitate endogenous EXPA17 from plant lysates, similar to the 89% success rate reported for synthetic antibodies against RNP complex proteins .

• Expression pattern correlation: Comparing immunolocalization patterns with transcriptional reporter lines (e.g., pEXPA17::GUS) to confirm that protein expression aligns with known transcript localization in lateral root primordia and overlaying tissues .

Documentation of these validation steps should be maintained to ensure confidence in experimental results obtained with the antibody.

How can EXPA17 antibodies be used to study lateral root development?

EXPA17 antibodies offer several powerful approaches to study lateral root development:

• Spatiotemporal expression analysis: Immunolocalization using EXPA17 antibodies can reveal the precise timing and location of EXPA17 protein accumulation during lateral root initiation, primordium formation, and emergence. This allows researchers to correlate EXPA17 activity with specific developmental stages of lateral root formation .

• Co-localization with cell wall modifications: Combining EXPA17 immunolocalization with cell wall staining techniques can demonstrate the relationship between EXPA17 presence and cell wall loosening in overlaying tissues, providing mechanistic insights into how expansins facilitate lateral root emergence .

• Protein-protein interaction studies: Immunoprecipitation with EXPA17 antibodies followed by mass spectrometry can identify protein complexes associated with EXPA17 during lateral root development, potentially revealing new components of the cell wall remodeling machinery.

• Hormone response dynamics: Using EXPA17 antibodies to track protein levels following auxin or cytokinin treatment can reveal post-transcriptional regulatory mechanisms that may not be evident from transcript analysis alone .

• Chromatin immunoprecipitation (ChIP) experiments: When studying transcriptional regulation of EXPA17, antibodies against transcription factors like LBD18 can be used in ChIP experiments to confirm direct binding to the EXPA17 promoter, as demonstrated for the EXPA14 promoter .

These approaches should be integrated with genetic studies using expa17 mutants and overexpression lines to establish causal relationships between EXPA17 function and phenotypic outcomes.

What protocols are recommended for EXPA17 immunolocalization in root tissues?

For successful immunolocalization of EXPA17 in Arabidopsis root tissues, the following protocol is recommended:

• Tissue fixation and embedding:

  • Fix freshly harvested root segments in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours at room temperature under vacuum

  • Gradually dehydrate tissues through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Infiltrate with a resin embedding medium suitable for immunohistochemistry

  • Section embedded tissue at 4-8 μm thickness

• Antigen retrieval and blocking:

  • Perform mild antigen retrieval using citrate buffer (pH 6.0) to expose epitopes

  • Block non-specific binding with 3% BSA, 0.1% Triton X-100 in PBS for 1 hour

• Primary antibody incubation:

  • Dilute EXPA17 antibody to appropriate concentration (typically 1:100 to 1:500) in blocking buffer

  • Incubate sections overnight at 4°C in a humid chamber

• Detection and visualization:

  • Use fluorophore-conjugated secondary antibodies matching the host species of the primary antibody

  • For dual labeling, combine with appropriate cell wall stains (e.g., Calcofluor White for cellulose)

  • Include controls: (i) sections from expa17 mutants, (ii) wild-type sections with primary antibody omitted

• Imaging optimization:

  • Use confocal laser scanning microscopy with appropriate filter settings

  • Employ optical sectioning to achieve high-resolution 3D visualization of EXPA17 distribution

  • Consider using roots expressing pEXPA17::EXPA17:GFP as positive controls for expression pattern comparison

This protocol should be optimized specifically for EXPA17 antibody concentration and incubation conditions based on signal-to-background ratio in initial experiments.

How can EXPA17 antibodies be used to investigate hormone-mediated regulation of cell wall remodeling?

EXPA17 antibodies enable sophisticated investigations into hormone-mediated regulation of cell wall remodeling through several advanced approaches:

• Quantitative immunoblotting for hormone response kinetics: Using EXPA17 antibodies for Western blot analysis after hormone treatments can reveal the timing of protein accumulation relative to transcript induction. This approach can uncover post-transcriptional regulation mechanisms, as EXPA17 transcript levels are known to increase in response to auxin treatment and LBD18 activation .

• Co-immunoprecipitation (Co-IP) of hormone-responsive complexes: EXPA17 antibodies can be used to immunoprecipitate protein complexes under different hormonal conditions, revealing dynamic changes in EXPA17-associated proteins that may function in coordinating hormone responses with cell wall modification.

• ChIP-qPCR for transcription factor binding dynamics: While not directly using EXPA17 antibodies, antibodies against transcription factors like LBD18 can be used in ChIP experiments to quantify binding to the EXPA17 promoter under various hormone treatments, connecting upstream signaling to EXPA17 expression .

• Proximity labeling approaches: Combining EXPA17 antibodies with proximity labeling techniques (BioID or APEX) can identify proteins in close proximity to EXPA17 within intact cells under different hormonal conditions, revealing context-specific interaction networks.

• Super-resolution microscopy of wall remodeling sites: Using EXPA17 antibodies with techniques like STORM or PALM microscopy can reveal nanoscale spatial relationships between EXPA17 and other cell wall components during hormone-induced loosening events.

These approaches help establish causal relationships between hormone signaling pathways, EXPA17 activity, and the biophysical changes in cell walls that facilitate plant development.

How can EXPA17 function be assessed in relation to cell wall biomechanics?

Assessing EXPA17's function in relation to cell wall biomechanics requires integrating antibody-based approaches with biophysical measurements:

• Correlative immunolocalization and mechanical mapping: Combining EXPA17 immunolocalization with atomic force microscopy (AFM) or Brillouin light scattering microscopy allows spatial correlation between EXPA17 presence and local cell wall mechanical properties . This approach can determine if regions with higher EXPA17 content show decreased cell wall stiffness, as observed with other expansins like EXPA15 .

• Mechano-optical contrast (MOC) measurements: Brillouin light scattering microscopy can be used to create spatial maps of cell wall stiffness in wild-type plants versus expa17 mutants, with immunolocalization of EXPA17 in adjacent sections to correlate protein presence with mechanical changes . This non-invasive technique allows for measuring mechanical properties in living tissues.

• Quantitative cell tomography: Holotomographic microscopy can measure refractive index as a proxy for cell wall density in regions with confirmed EXPA17 expression (via immunolocalization), allowing researchers to determine if EXPA17 affects the mass content of cell walls during loosening .

• In vitro extension assays with immunodepleted extracts: Cell wall protein extracts can be immunodepleted of EXPA17 using specific antibodies, then tested in in vitro extension assays to quantify the contribution of EXPA17 to wall loosening activity compared to complete extracts.

• Creep rate measurements: Measuring the creep rate (time-dependent extension under constant force) of isolated cell walls treated with purified EXPA17 protein can directly quantify its wall loosening activity, which can then be correlated with in vivo localization patterns determined by immunohistochemistry.

These combined approaches provide a comprehensive understanding of how EXPA17 modifies cell wall properties in the context of lateral root development.

What methodological considerations are important when using EXPA17 antibodies in protein-protein interaction studies?

When using EXPA17 antibodies for protein-protein interaction studies, several methodological considerations are critical:

• Crosslinking optimization for cell wall proteins: Due to EXPA17's association with the cell wall, crosslinking conditions must be carefully optimized. Formaldehyde concentrations of 1-2% for 10-15 minutes are typically suitable, but pilot experiments should determine optimal conditions that preserve interactions without creating non-specific aggregates.

• Extraction buffer composition: Cell wall-associated proteins require specialized extraction conditions. Buffers containing 1M NaCl or 4M LiCl can help solubilize ionically-bound cell wall proteins, while maintaining conditions that preserve protein-protein interactions. Adding protease inhibitors and appropriate detergents (0.1-0.5% Triton X-100 or NP-40) is essential.

• Pre-clearing strategy: Plant extracts contain compounds that can cause non-specific binding. Pre-clearing lysates with protein A/G beads and non-immune IgG from the same species as the EXPA17 antibody helps reduce background.

• Validation with reciprocal co-IPs: Any interaction identified by EXPA17 immunoprecipitation should be confirmed through reciprocal co-IP experiments using antibodies against the putative interacting partners.

• Controls for specificity: Several controls are essential:

  • Immunoprecipitation from expa17 mutant tissues to identify non-specific binding

  • Use of pre-immune serum or isotype-matched control antibodies

  • Competition experiments with excess recombinant EXPA17 protein

• Consideration of detergent effects: Different detergents can disrupt certain types of protein-protein interactions. Testing multiple detergent conditions (from milder options like digitonin to stronger ones like SDS) can help determine the stability of identified interactions.

• Proximity-dependent techniques: For transient or weak interactions, consider using antibodies in proximity-dependent approaches like proximity ligation assay (PLA), which can detect proteins within 40 nm of each other in fixed tissue.

Implementing these considerations ensures that interactions identified using EXPA17 antibodies reflect biologically relevant associations rather than experimental artifacts.

What are common challenges when working with EXPA17 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with EXPA17 antibodies, each requiring specific troubleshooting approaches:

• High background in immunolocalization:

  • Cause: Non-specific binding, insufficient blocking, or cross-reactivity with other expansins

  • Solution: Increase blocking time/concentration (5% BSA instead of 3%), use additional blocking agents (5% normal serum from secondary antibody host species), include 0.05-0.1% Tween-20 in wash buffers, and optimize antibody dilution through titration experiments

• Weak or absent signal in Western blots:

  • Cause: Inefficient extraction of cell wall-associated EXPA17, protein degradation, or low endogenous expression

  • Solution: Use specialized extraction buffers containing urea (4-8M) or high salt (1M NaCl) to solubilize cell wall proteins, add additional protease inhibitors, increase protein loading, and extend primary antibody incubation time (overnight at 4°C)

• Cross-reactivity with other expansins:

  • Cause: Antibody recognizes conserved epitopes present in multiple expansin family members

  • Solution: Pre-absorb antibody with recombinant proteins of closely related expansins like EXPA1, EXPA10, EXPA14, and EXPA15 , or develop new antibodies against unique peptide regions of EXPA17

• Poor immunoprecipitation efficiency:

  • Cause: Insufficient antibody affinity or accessibility of epitopes in native conditions

  • Solution: Optimize antibody-to-bead ratio, extend incubation time, use gentle rotation instead of shaking, test different lysis/IP buffers with varying salt and detergent concentrations

• Inconsistent results between experiments:

  • Cause: Variation in plant growth conditions affecting EXPA17 expression levels

  • Solution: Standardize growth conditions rigorously, include internal controls in each experiment, and normalize results to housekeeping proteins

• Interference from plant secondary metabolites:

  • Cause: Polyphenols and other compounds can interfere with antibody binding

  • Solution: Add polyvinylpyrrolidone (PVP) or polyvinylpolypyrrolidone (PVPP) to extraction buffers, increase concentration of reducing agents like DTT or β-mercaptoethanol

Documenting optimization steps systematically and sharing these details in publications will help advance the field by providing reliable protocols for EXPA17 research.

How can EXPA17 antibody performance be optimized for different experimental applications?

Optimizing EXPA17 antibody performance requires application-specific adjustments:

• For Western blotting:

  • Sample preparation: Extract proteins using buffer containing 4M urea, 100mM Tris-HCl (pH 8.0), 1M NaCl, 5mM EDTA, 1% Triton X-100, and protease inhibitors

  • Transfer conditions: Use PVDF membranes (rather than nitrocellulose) for better protein retention

  • Blocking: Block with 5% non-fat dry milk in TBST for 2 hours at room temperature

  • Antibody dilution: Start with 1:1000 dilution in 3% BSA in TBST, incubate overnight at 4°C

  • Detection enhancement: Consider using high-sensitivity ECL substrates or signal amplification systems

• For immunohistochemistry/immunofluorescence:

  • Fixation: Test both aldehydes (4% paraformaldehyde) and alcohol-based fixatives to determine optimal epitope preservation

  • Antigen retrieval: Perform heat-induced epitope retrieval in sodium citrate buffer (pH 6.0)

  • Antibody dilution: Begin with 1:200 dilution, then optimize based on signal-to-noise ratio

  • Signal amplification: Consider tyramide signal amplification for low-abundance detection

  • Background reduction: Include 0.1% Triton X-100 and 5% normal serum in antibody diluent

• For immunoprecipitation:

  • Antibody coupling: Covalently couple antibodies to beads to prevent heavy/light chain interference

  • Pre-clearing: Pre-clear lysates with protein A/G beads for 1 hour before adding EXPA17 antibody

  • Buffer optimization: Test different salt concentrations (150-500mM NaCl) to find the optimal stringency

  • Incubation conditions: Extend incubation to overnight at 4°C with gentle rotation

  • Elution strategies: Compare harsh (SDS, low pH) vs. gentle (peptide competition) elution methods

• For chromatin immunoprecipitation (ChIP):

  • Crosslinking: Optimize formaldehyde concentration (1-3%) and crosslinking time (5-15 minutes)

  • Sonication: Adjust sonication conditions to achieve 200-500 bp chromatin fragments

  • Antibody amount: Use 2-5 μg of antibody per ChIP reaction, optimizing through titration

  • Controls: Include IgG control and input samples in each experiment

  • Quantification: Use qPCR with primers spanning known binding regions and negative control regions

Application-specific optimization should be conducted systematically, changing one variable at a time and documenting outcomes to establish robust protocols for EXPA17 research.

How might new antibody technologies advance EXPA17 research?

Emerging antibody technologies offer significant potential to advance EXPA17 research:

• Single-domain antibodies (nanobodies): These smaller antibody fragments derived from camelid heavy-chain antibodies can access restricted epitopes that might be inaccessible to conventional antibodies, potentially improving detection of EXPA17 in its native cell wall environment. Their small size (~15 kDa) allows better penetration into dense cell wall structures.

• Proximity-labeling antibody fusion proteins: By generating fusions of EXPA17 antibodies with enzymes like BioID2 or TurboID, researchers can identify proteins in close proximity to EXPA17 in living cells. This approach could reveal transient interaction partners involved in cell wall loosening that might be missed in conventional co-immunoprecipitation approaches.

• Bispecific antibodies: Engineered bispecific antibodies targeting both EXPA17 and other cell wall remodeling enzymes could enable precise co-localization studies and potentially reveal functional interactions between different components of the cell wall modification machinery.

• Antibody-based optogenetic tools: Antibody fragments fused to light-sensitive domains could allow for spatial and temporal control of EXPA17 activity or localization, enabling precise manipulation of cell wall loosening during lateral root development.

• Intrabodies with conditional stability domains: Developing EXPA17-specific antibody fragments that function intracellularly (intrabodies) with conditional stability domains would allow for rapid, inducible inhibition of EXPA17 function without genetic modification of the plant.

• High-throughput synthetic antibody pipelines: As demonstrated for other proteins, high-throughput methods for generating synthetic antibodies could produce panels of EXPA17 antibodies recognizing different epitopes, enabling more comprehensive analysis of EXPA17 function .

These technological advances would significantly enhance our ability to study EXPA17's dynamic role in cell wall remodeling during plant development.

What are promising research directions for EXPA17 antibodies in understanding plant development under stress conditions?

EXPA17 antibodies offer several promising research directions for understanding plant development under stress conditions:

• Spatiotemporal dynamics during abiotic stress: Using EXPA17 antibodies to track protein localization and abundance changes during drought, salinity, or temperature stress could reveal how stress conditions modify lateral root development through altered cell wall remodeling activities. Comparative immunolocalization studies between stressed and non-stressed plants would identify specific tissues where EXPA17 activity is modified.

• Post-translational modification analysis: Developing antibodies specific for post-translationally modified forms of EXPA17 (phosphorylated, glycosylated, etc.) would enable studies of how stress conditions might regulate EXPA17 activity through these modifications rather than just affecting expression levels.

• Hormone crosstalk investigation: Given that EXPA17 is regulated by auxin , examining how stress-related hormones (ABA, ethylene, jasmonic acid) affect EXPA17 protein levels and localization could reveal mechanisms of growth-defense tradeoffs during stress responses.

• Cell wall composition correlation: Combining EXPA17 immunolocalization with glycome profiling of cell walls under stress conditions could establish relationships between EXPA17 activity and specific changes in cell wall polymer composition and cross-linking.

• Root system architecture regulation: Using EXPA17 antibodies to study lateral root development under different nutritional stresses could reveal how plants modulate their root architecture to optimize resource acquisition through targeted cell wall remodeling.

• Interspecies comparative analysis: Developing antibodies that recognize EXPA17 homologs across multiple plant species would enable comparative studies of how this mechanism of stress adaptation has evolved in species with different stress tolerance levels.

These research directions would significantly advance our understanding of how plants modulate development through cell wall remodeling in response to environmental challenges.

What best practices should researchers follow when incorporating EXPA17 antibodies in their experimental workflows?

Researchers should adopt the following best practices when incorporating EXPA17 antibodies into their experimental workflows:

• Comprehensive validation: Before using an EXPA17 antibody for main experiments, validate its specificity using multiple approaches including Western blot, immunoprecipitation, and immunolocalization, comparing signals between wild-type and expa17 mutant plants . Document validation results for reference and publication.

• Application-specific optimization: Optimize antibody concentration, incubation conditions, and buffer compositions separately for each application (Western blotting, immunolocalization, immunoprecipitation), as optimal conditions will differ significantly between techniques.

• Consistent plant growth conditions: Standardize plant growth conditions rigorously, as EXPA17 expression is hormone-responsive and environmentally sensitive . Document growth parameters in detail to ensure reproducibility.

• Multiple biological replicates: Always include at least three biological replicates in experiments, as EXPA17 expression can vary between individual plants even under controlled conditions.

• Appropriate controls: Include all necessary controls in each experiment:

  • Negative controls (expa17 mutants, pre-immune serum)

  • Positive controls (tissues known to express EXPA17)

  • Technical controls (loading controls for Western blots, reference genes for qPCR)

• Complementary approaches: Combine antibody-based detection with other methods, such as fluorescent protein fusions or promoter-reporter constructs, to cross-validate findings .

• Careful data interpretation: When interpreting results, consider the limitations of antibody-based detection, including potential cross-reactivity with other expansins, which share significant sequence homology .

• Detailed methodology reporting: When publishing, provide comprehensive methodological details including antibody source, validation evidence, dilutions, and incubation conditions to facilitate reproducibility.

Following these best practices will maximize data quality and reproducibility in EXPA17 research, advancing our understanding of cell wall remodeling in plant development.

What key considerations should guide researchers in selecting or developing EXPA17 antibodies for specific research questions?

When selecting or developing EXPA17 antibodies for specific research questions, researchers should consider:

• Research question alignment: Clearly define what aspect of EXPA17 biology you need to investigate:

  • For protein localization studies, antibodies recognizing native protein conformations are essential

  • For quantification studies, antibodies with linear epitope recognition work better for Western blotting

  • For interaction studies, antibodies binding regions away from interaction surfaces are preferable

• Epitope selection strategy: Consider the unique challenges of EXPA17 as a target:

  • Target regions unique to EXPA17 that differentiate it from other expansins (EXPA1, EXPA10, EXPA14, EXPA15)

  • Avoid the conserved central domain shared among expansins

  • Consider using computational tools to identify surface-exposed regions likely to be immunogenic

• Antibody format requirements:

  • Monoclonal antibodies offer consistency across experiments and batches

  • Polyclonal antibodies may provide better sensitivity by recognizing multiple epitopes

  • Recombinant antibodies allow for protein engineering and consistent renewable supply

  • Consider species origin based on planned applications (avoid rabbit antibodies if studying rabbit tissues)

• Validation stringency needs:

  • For exploratory studies, basic validation may suffice

  • For mechanistic studies or publication in high-impact journals, comprehensive validation including genetic controls (expa17 mutants) is essential

  • For quantitative studies, standard curves with recombinant EXPA17 are necessary

• Technical application requirements:

  • For immunofluorescence in plant tissues, antibodies with high affinity and specificity are required

  • For chromatin immunoprecipitation, antibodies must function under crosslinking conditions

  • For co-immunoprecipitation, antibodies must not disrupt protein-protein interactions

• Scale and reproducibility needs:

  • Consider the long-term availability of antibodies for extended research programs

  • For high-throughput studies, synthetic antibody approaches may provide better reproducibility