EXPA15 Antibody

What is EXPA15 and what is its primary function in plant biology?

EXPA15 is a member of the alpha-expansin family in Arabidopsis thaliana (AtEXPA15, encoded by At2g03090). Expansins facilitate cell expansion through mediating pH-dependent cell wall loosening, which is crucial for plant growth and development. EXPA15 specifically contributes to the regulation of biomechanical cell wall properties in root tissues . Unlike some other expansins that decrease cell wall stiffness, EXPA15 has been associated with localization patterns that overlap with regions of higher cell wall stiffness as measured by Brillouin light scattering microscopy .

How is EXPA15 expression regulated in plant cells?

EXPA15 expression is primarily regulated by plant hormones, particularly cytokinins. Research has demonstrated that EXPA15 is strongly upregulated in response to exogenous cytokinin application . This upregulation appears to be mediated through cytokinin-activated type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), suggesting EXPA15 may be a direct target of cytokinin signaling pathways . The induction of EXPA15 by cytokinins is notably reduced in lines overexpressing ARR7 (a type-A ARR and negative regulator of cytokinin signaling) .

What is the subcellular and tissue localization pattern of EXPA15?

EXPA15 exhibits a distinctive localization pattern that has been visualized using EXPA15:mCherry fusion proteins. It localizes to the root apical meristem (RAM) epidermis and emerging lateral roots in a relatively uniform pattern . In deeper tissues, EXPA15 shows a "spotty" pattern at three-cell boundaries in the cortex/endodermis. As distance increases from the meristematic zone, EXPA15 relocalizes into deeper vasculature layers with a more homogeneous distribution . This specific localization pattern distinguishes EXPA15 from other expansins like EXPA1, EXPA10, and EXPA14, which display different tissue and subcellular distribution patterns.

What approaches can be used to develop specific antibodies against EXPA15?

Developing effective antibodies against EXPA15 requires strategic planning due to potential cross-reactivity with other expansin family members. The recommended approach includes:

• Peptide selection: Identify unique epitopes in the EXPA15 sequence that have minimal homology with other expansin family members. The C-terminal region often provides higher specificity.

• Recombinant protein expression: Express the full-length or partial EXPA15 protein in bacterial systems (like E. coli) with appropriate tags for purification.

• Immunization strategy: Use either synthetic peptides or recombinant proteins for immunization of host animals (typically rabbits for polyclonal or mice for monoclonal antibodies).

• Antibody purification: Employ affinity purification against the immunizing antigen to enhance specificity.

• Cross-adsorption: Remove cross-reactive antibodies by pre-adsorption against closely related expansins.

What methods are most effective for validating the specificity of EXPA15 antibodies?

A multi-tiered approach to validation ensures the highest specificity:

• Western blotting against recombinant proteins: Test against purified EXPA15 and related expansins (especially EXPA1, EXPA10, EXPA14) to evaluate cross-reactivity.

• Knockout/knockdown controls: Validate using samples from EXPA15 knockout or knockdown plants compared to wild-type.

• Overexpression verification: Test in systems with EXPA15 overexpression to confirm increased signal.

• Epitope competition assays: Pre-incubate antibodies with the immunizing peptide to demonstrate signal specificity.

• Immunoprecipitation followed by mass spectrometry: Confirm that the antibody is pulling down EXPA15 rather than other proteins.

• Immunohistochemistry pattern comparison: Compare antibody localization pattern with EXPA15:mCherry fusion protein localization data to verify consistency .

How can EXPA15 antibodies be optimized for immunohistochemistry in plant tissues?

Optimizing EXPA15 antibodies for immunohistochemistry requires addressing several plant-specific challenges:

• Fixation protocol optimization: Test both aldehyde-based (4% paraformaldehyde) and alcohol-based fixatives to determine which best preserves EXPA15 epitopes while maintaining tissue architecture.

• Cell wall permeabilization: Include additional enzymatic digestion steps (using cellulase and pectinase) to improve antibody penetration through plant cell walls.

• Blocking optimization: Use a combination of bovine serum albumin (3-5%) and normal serum (5-10%) from the same species as the secondary antibody to reduce background.

• Signal amplification: Consider tyramide signal amplification for low-abundance EXPA15 detection.

• Antibody concentration titration: Test a range of primary antibody concentrations (typically 1:100 to 1:1000) to determine optimal signal-to-noise ratio.

• Extended incubation: Allow longer primary antibody incubation (overnight at 4°C to 48 hours) to improve penetration in plant tissues.

• Clearing techniques: Implement tissue clearing methods (ClearSee or modified Scale) to improve imaging depth and resolution.

What are the recommended protocols for using EXPA15 antibodies in Western blotting?

For optimal Western blotting results with EXPA15 antibodies:

• Sample preparation:

  • Extract proteins using a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

  • Include 1% polyvinylpolypyrrolidone (PVPP) to remove plant phenolic compounds

  • Concentrate cell wall proteins using ammonium sulfate precipitation if necessary

• Gel electrophoresis conditions:

  • Use 12-15% SDS-PAGE gels for optimal resolution of EXPA15 (approximately 25-30 kDa)

  • Include positive controls (recombinant EXPA15) and size markers

• Transfer conditions:

  • Transfer to PVDF membranes at 25V overnight at 4°C for complete transfer

  • Verify transfer using reversible staining (Ponceau S)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with primary antibody (1:1000 dilution) overnight at 4°C

  • Wash 3x15 minutes with TBST

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature

• Detection optimization:

  • Use enhanced chemiluminescence substrate optimized for low-abundance proteins

  • Consider longer exposure times (up to 10 minutes) for weak signals

How can mutational analysis approaches be applied to study EXPA15 epitopes and improve antibody specificity?

Mutational analysis can significantly enhance EXPA15 antibody development through systematic epitope mapping and specificity refinement:

• Deep mutational scanning: This technique, similar to that applied for antibodies against epidermal growth factor receptor , can be adapted for EXPA15. By creating libraries with comprehensive point mutations in key EXPA15 regions and analyzing antibody binding, researchers can identify critical residues for recognition.

• Alanine scanning mutagenesis: Systematically replace surface-exposed residues with alanine to identify which amino acids are essential for antibody recognition.

• Domain swapping: Exchange domains between EXPA15 and closely related expansins (EXPA1, EXPA10, EXPA14) to identify regions that confer specificity.

• Molecular display technologies: Utilize yeast or phage display systems to present EXPA15 variants and select for high-affinity binders through successive rounds of selection.

• Computational epitope prediction: Employ in silico approaches to predict immunogenic regions unique to EXPA15 compared to other expansins before experimental validation.

This comprehensive mutational approach can identify epitopes that maximize specificity, allowing researchers to develop antibodies that can distinguish between closely related expansin family members .

What strategies can be implemented to investigate EXPA15 interactions with other proteins using antibody-based approaches?

Several advanced techniques can be employed to study EXPA15 protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use EXPA15 antibodies conjugated to magnetic or agarose beads

  • Perform pulldowns under native conditions to preserve protein-protein interactions

  • Analyze precipitated complexes by mass spectrometry to identify interacting partners

  • Cross-validate findings with reciprocal Co-IPs using antibodies against putative interactors

• Proximity labeling coupled with immunoprecipitation:

  • Generate EXPA15 fusions with BioID or APEX2 proximity labeling enzymes

  • Express these constructs in plant cells to biotinylate proximal proteins

  • Use EXPA15 antibodies to confirm proper localization of fusion proteins

  • Purify biotinylated proteins and identify by mass spectrometry

• Förster Resonance Energy Transfer (FRET):

  • Label EXPA15 antibodies (or Fab fragments) with donor fluorophores

  • Label putative interacting proteins with acceptor fluorophores

  • Measure energy transfer to confirm close proximity (<10 nm) in living cells

• Antibody-based protein complex purification:

  • Use crosslinking agents to stabilize transient interactions before extraction

  • Apply EXPA15 antibodies for gentle immunoaffinity purification

  • Analyze complexes using blue native PAGE or size exclusion chromatography combined with Western blotting

• Super-resolution microscopy with dual immunolabeling:

  • Use EXPA15 antibodies in combination with antibodies against cell wall remodeling enzymes

  • Apply techniques like STORM or PALM to achieve nanometer resolution

  • Quantify colocalization statistics to infer potential functional relationships

How can non-specific binding be reduced when using EXPA15 antibodies in plant tissues?

Non-specific binding is a common challenge when working with plant tissues. To minimize this issue with EXPA15 antibodies:

• Antibody purification refinement:

  • Implement additional purification steps like negative selection against plant extracts from EXPA15 knockout lines

  • Use affinity chromatography with immobilized recombinant EXPA15 for antibody enrichment

  • Consider subtractive purification against closely related expansins (EXPA1, EXPA10, EXPA14)

• Optimization of blocking protocols:

  • Test plant-specific blocking agents like 5% non-fat milk supplemented with 1% plant-derived protein extracts

  • Include 0.1-0.3% Triton X-100 in blocking buffers to reduce hydrophobic interactions

  • Add 10-20 mM glycine to quench any remaining aldehyde groups from fixation

• Pre-adsorption protocols:

  • Incubate antibodies with acetone powder prepared from EXPA15-deficient plant tissues

  • Pre-clear with a mixture of recombinant EXPA1, EXPA10, and EXPA14 proteins to remove cross-reactive antibodies

  • Test multiple temperatures (4°C, room temperature) and times (1-24 hours) for pre-adsorption

• Sample preparation modifications:

  • Increase washing steps and durations (4-5 washes of 15 minutes each)

  • Include 0.05-0.1% SDS in wash buffers for immunoblotting applications

  • Consider antigen retrieval methods (citrate buffer pH 6.0, microwave treatment) for fixed samples

What approaches can resolve contradictory results between EXPA15 antibody labeling and fusion protein localization data?

When faced with discrepancies between antibody labeling patterns and fusion protein localization:

• Systematic comparative analysis:

  • Generate side-by-side comparisons in identical tissues and developmental stages

  • Document all differences in subcellular localization patterns

  • Quantify signal intensities in different cell types and compartments

• Validation with multiple methodologies:

  • Confirm EXPA15 localization using complementary approaches:

    • Antibody labeling

    • Fluorescent protein fusions (both N- and C-terminal)

    • mRNA in situ hybridization

    • Mass spectrometry of isolated cell walls/organelles

• Technical factor assessment:

  • Evaluate whether fusion proteins could disrupt EXPA15 targeting signals

  • Test multiple fixation protocols that might differentially preserve epitopes

  • Examine whether antibodies access all cellular compartments equally

  • Consider the effects of overexpression on localization patterns

• Biological explanation investigation:

  • Assess whether different detection methods capture distinct EXPA15 populations

  • Test whether EXPA15 localization is dynamic and condition-dependent

  • Examine potential post-translational modifications that might affect antibody recognition

  • Use pulse-chase experiments to track EXPA15 trafficking and turnover

How can EXPA15 antibodies be applied to investigate the relationship between cell wall stiffness and root development?

EXPA15 antibodies can be powerful tools for investigating the mechanistic relationship between cell wall properties and root development:

• Correlative microscopy approaches:

  • Combine immunolocalization of EXPA15 with Brillouin light scattering microscopy to directly correlate protein localization with cell wall stiffness measurements in specific root zones

  • Overlay EXPA15 distribution maps with atomic force microscopy (AFM) measurements of Young's modulus in matched samples

  • Track dynamic changes in EXPA15 localization during gravitropic responses or mechanical stress

• Perturbation experiments:

  • Apply EXPA15-blocking antibodies directly to growing roots to inhibit function in specific regions

  • Use inducible EXPA15 overexpression or knockdown systems combined with immunolabeling to track acute changes in cell wall properties

  • Develop synthetic cell wall-anchoring peptides based on EXPA15 binding sites to competitively inhibit its function

• Hormone response analysis:

  • Map cytokinin response gradients in relation to EXPA15 distribution using reporter lines

  • Apply exogenous hormones and track subsequent changes in EXPA15 localization and cell wall properties

  • Combine with mechanical measurements to test the hypothesis that EXPA15 upregulation by cytokinins leads to local changes in cell wall stiffness

• Quantitative imaging analysis:

  • Develop computational pipelines to quantify EXPA15 abundance in different cell types and correlate with growth rates

  • Implement machine learning approaches to classify cell wall properties based on EXPA15 immunolabeling patterns

  • Create predictor models for root development based on EXPA15 distribution and cell wall mechanics

What are the cutting-edge approaches for studying EXPA15 function in living plant tissues using antibody-derived tools?

Recent technological advances offer new possibilities for studying EXPA15 in vivo:

• Nanobody and intrabody development:

  • Generate single-domain antibodies (nanobodies) against EXPA15 from camelid immunizations

  • Express these nanobodies fused to fluorescent proteins in planta to track endogenous EXPA15

  • Create "intrabodies" that can be expressed inside cells to modulate EXPA15 function without genetic modification

• CRISPR-based tagging for endogenous labeling:

  • Use CRISPR/Cas9 to insert small epitope tags into the endogenous EXPA15 gene

  • Validate tag function using established anti-tag antibodies

  • Compare localization patterns of tagged endogenous EXPA15 with antibody staining of the native protein

• Optogenetic tools combined with antibody validation:

  • Develop light-inducible EXPA15 expression or degradation systems

  • Use antibodies to quantify protein dynamics following optogenetic activation

  • Create optogenetic systems to control EXPA15 localization and verify with immunolabeling

• Expansion microscopy applications:

  • Adapt expansion microscopy protocols for plant tissues

  • Use EXPA15 antibodies for super-resolution imaging of physically expanded samples

  • Map nanoscale distribution of EXPA15 in relation to other cell wall components

How should researchers interpret differences in EXPA15 antibody signal patterns between different root zones?

When analyzing variable EXPA15 antibody signals across root zones, consider this interpretation framework:

Proper interpretation should:

• Compare patterns with EXPA15:mCherry fusion protein localization data

• Account for cell type-specific differences in antibody penetration

• Consider dynamic changes during development

• Correlate with known cytokinin response patterns

• Integrate with cell wall stiffness measurements from complementary techniques

What statistical approaches are most appropriate for quantifying EXPA15 distribution from immunohistochemistry data?

Robust quantification of EXPA15 immunolabeling requires sophisticated statistical approaches:

• Signal intensity normalization methods:

  • Use internal reference standards (constitutively expressed proteins) for relative quantification

  • Apply ratiometric analysis against cell wall counterstains

  • Implement tissue-specific autofluorescence correction algorithms

• Spatial distribution analysis:

  • Employ Ripley's K-function to analyze clustering patterns

  • Use nearest neighbor distance analysis for pattern recognition

  • Apply Moran's I test to quantify spatial autocorrelation of signal

• Machine learning approaches:

  • Train neural networks to classify EXPA15 distribution patterns

  • Implement segmentation algorithms to distinguish cell-specific localization

  • Use transfer learning from established cell wall protein datasets

• Comparative statistical tests:

  • Apply ANOVA with post-hoc tests for multi-zone comparisons

  • Use Mann-Whitney U test for non-parametric comparisons between sample pairs

  • Implement mixed-effects models to account for biological and technical variability

• Meta-analysis framework:

  • Develop standardized scoring systems to integrate results across experiments

  • Create confidence metrics based on signal-to-noise ratios

  • Establish minimum reporting standards for immunolocalization studies

These approaches should be selected based on specific experimental designs and data characteristics, with careful attention to statistical assumptions and appropriate controls.