EXPA13 Antibody

What is EXPA13 and what cellular functions does it serve?

EXPA13 is a member of the α-expansin family in plants that plays crucial roles in cell wall loosening and expansion. Research indicates EXPA13 is specifically involved in seed imbibition processes and is functionally associated with dormancy release mechanisms. EXPA13 expression is significantly induced during seed imbibition and is regulated by the MKK3-MPK7 signaling cascade. Overexpression of EXPA13 has been demonstrated to attenuate seed dormancy, highlighting its role in embryo cell expansion during seed germination .

How are EXPA13 antibodies typically generated?

EXPA13 antibodies are typically generated using synthetic peptides derived from unique regions of the protein sequence. Similar to the approach used for other expansin antibodies, researchers select antigenic regions with high specificity (often 15-20 amino acids in length) for immunization in rabbits or other host animals. For instance, antibodies against the related expansin OsEXPA10 were generated using a synthetic peptide C-VPKEWDFGKTYTGKQFLL (positions 240-257) . When selecting peptide sequences for EXPA13 antibody production, researchers must carefully analyze unique regions that minimize cross-reactivity with other expansin family members, particularly considering the high sequence similarity between some expansin proteins.

What are the typical applications of EXPA13 antibodies in plant research?

EXPA13 antibodies are valuable tools for:

• Immunolocalization studies to determine tissue and cellular distribution patterns

• Western blot analysis to quantify protein expression levels

• Immunoprecipitation to study protein-protein interactions

• Chromatin immunoprecipitation (ChIP) when investigating transcription factors that regulate EXPA13

• Monitoring changes in EXPA13 protein abundance during developmental processes or in response to environmental stimuli

Similar to other expansin antibodies, they can be applied to correlate protein expression with tissue-specific functions, particularly in seed tissues where EXPA13 has been shown to be functionally important for dormancy release .

What protocols should be followed for effective immunolocalization of EXPA13 in plant tissues?

For effective immunolocalization of EXPA13 in plant tissues, researchers should follow this optimized protocol:

• Tissue fixation: Fix tissues in 4% paraformaldehyde in PBS for 2-4 hours at room temperature or overnight at 4°C.

• Embedding and sectioning: Embed in paraffin or resin and prepare sections of 5-10 μm thickness.

• Antigen retrieval: Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-15 minutes to expose epitopes that may be masked during fixation.

• Blocking: Block with 2-3% BSA in PBS containing 0.1% Triton X-100 for 1 hour to reduce non-specific binding.

• Primary antibody incubation: Apply diluted EXPA13 antibody (typically 1:100 to 1:500) and incubate overnight at 4°C.

• Washing: Wash 3-5 times with PBS containing 0.1% Tween-20.

• Secondary antibody: Apply fluorescently labeled secondary antibody (e.g., Alexa Fluor 555 goat anti-rabbit IgG) at 1:200-1:500 dilution for 1-2 hours at room temperature.

• Counterstaining: Use DAPI (1 μg/mL) for nucleus visualization.

• Mounting and imaging: Mount with anti-fade medium and observe using confocal laser scanning microscopy.

This approach is similar to the immunostaining protocol used for OsEXPA10 localization studies, which successfully revealed tissue-specific distribution patterns in root tissues .

How can I optimize Western blot conditions for EXPA13 detection?

Optimizing Western blot conditions for EXPA13 detection requires careful consideration of several parameters:

• Protein extraction: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and protease inhibitor cocktail. For plant tissues, add 1% PVPP to remove phenolic compounds.

• Sample preparation: Heat samples at 70°C (rather than boiling) for 10 minutes in Laemmli buffer to minimize protein aggregation.

• Gel percentage and running conditions:

  Protein Size	Gel Percentage	Running Voltage	Running Time
  EXPA13 (~25-30 kDa)	12-15%	100-120V	1-1.5 hours

• Transfer conditions: Use semi-dry transfer at 15V for 30 minutes or wet transfer at 30V overnight at 4°C.

• Blocking: 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Antibody dilution: Primary antibody (1:1000 to 1:3000) in 1% milk-TBST, incubate overnight at 4°C; secondary HRP-conjugated antibody (1:5000 to 1:10000) for 1 hour at room temperature.

• Detection: Enhanced chemiluminescence (ECL) with exposure times optimized based on signal intensity.

This optimization approach ensures specific detection of EXPA13 while minimizing background and cross-reactivity with other expansin family members .

What controls should be included when working with EXPA13 antibodies?

When working with EXPA13 antibodies, comprehensive controls are essential to ensure experimental validity:

Positive controls:

• Recombinant EXPA13 protein or overexpression lines

• Tissues known to express EXPA13 (e.g., imbibing seeds based on expression data)

• Samples treated with conditions known to upregulate EXPA13 (e.g., GA treatment in seeds)

Negative controls:

• EXPA13 knockout or knockdown lines (critical for antibody validation)

• Pre-immune serum in place of primary antibody

• Primary antibody omission

• Blocking peptide competition assay (incubating antibody with the immunizing peptide before application)

• Tissues known not to express EXPA13

The approach of using genetic knockout lines has proven effective for validating antibody specificity, as demonstrated in OsEXPA10 studies where immunostaining signals were absent in knockout lines, confirming antibody specificity .

How can I distinguish between different expansin family members when using antibodies?

Distinguishing between expansin family members requires careful antibody design and validation due to sequence homology. Implement these strategies:

• Epitope selection: Choose peptide epitopes from highly divergent regions:

  • C-terminal regions often show greater sequence diversity

  • Avoid conserved domains like the HFD motif (histidine-phenylalanine-aspartate) that is present in most expansins

• Cross-reactivity testing: Test antibody against recombinant proteins of multiple expansin family members, particularly EXPA1, EXPA3, EXPA8, EXPA9, EXPA10, and EXPA15, which are often co-expressed with EXPA13 .

• Knockout validation: Validate in EXPA13 knockout lines to confirm signal absence, while checking signal presence in knockouts of other expansin genes.

• Western blot optimization: Use high-resolution SDS-PAGE (15%) to separate similarly sized expansins.

• Immunoprecipitation followed by mass spectrometry: Confirm antibody specificity by identifying pulled-down proteins.

The approach used for OsEXPA10 antibody validation demonstrates the effectiveness of using genetic knockout lines to confirm antibody specificity in plant tissues .

How can I study the regulation of EXPA13 by protein kinase signaling pathways?

Studying the regulation of EXPA13 by protein kinase signaling pathways, particularly the MKK3-MPK7 cascade, requires a multi-faceted approach:

• Kinase activity assays:

  • Perform in vitro kinase assays with recombinant MKK3/MPK7 and potential transcription factors (like ERF4) that regulate EXPA13

  • Use phosphorylation-specific antibodies to monitor kinase activation in vivo

• Gene expression analysis:

  • Compare EXPA13 expression in wild-type vs. kinase mutants (mkk3, mpk7) using qRT-PCR

  • Analyze expression changes in response to dormancy-breaking treatments in different genetic backgrounds

• Chromatin immunoprecipitation (ChIP):

  • Identify transcription factors (like ERF4) binding to the EXPA13 promoter

  • Compare binding in phosphorylated vs. non-phosphorylated states of the transcription factor

• Protein-protein interactions:

  • Use co-immunoprecipitation with EXPA13 antibodies to identify interacting kinases or substrates

  • Perform yeast two-hybrid or BiFC analyses to confirm direct interactions

• Genetic approaches:

  • Create transgenic lines with EXPA13 expression in kinase mutant backgrounds

  • Analyze phenotypic rescue to establish functional relationships

Research has demonstrated that the MKK3-MPK7 module regulates multiple expansins including EXPA13 during seed imbibition, and overexpression of EXPA13 can rescue the dormancy-enhancing phenotype of mpk7 mutants, confirming the functional relationship between these signaling components .

What methodologies can be used to correlate EXPA13 protein levels with cell wall modifications?

Correlating EXPA13 protein levels with cell wall modifications requires integrating multiple analytical approaches:

• Quantitative protein analysis:

  • Western blotting with EXPA13 antibodies to quantify protein levels

  • Mass spectrometry-based proteomics for absolute quantification

• Cell wall mechanical properties:

  • Atomic force microscopy (AFM) to measure cell wall elasticity and extensibility

  • Mechanical extensometer assays to measure wall creep under acidic conditions

  • Stress-relaxation analyses of isolated cell walls

• Cell wall composition analysis:

  • Immunolabeling of cell wall components (pectins, xyloglucans, celluloses)

  • FTIR spectroscopy to detect chemical modifications

  • Comprehensive Microarray Polymer Profiling (CoMPP) for high-throughput analysis

• Microscopy techniques:

  • Transmission electron microscopy to visualize cell wall ultrastructure

  • Co-localization of EXPA13 with cell wall components using double immunolabeling

  • Live-cell imaging with fluorescently tagged EXPA13 to monitor dynamics

• Genetic approaches:

  • Compare cell wall properties in EXPA13 overexpression vs. knockout lines

  • Analyze EXPA13 distribution and cell wall modifications during specific developmental processes

This multi-technique approach provides a comprehensive understanding of how EXPA13 protein abundance correlates with specific cell wall modifications, similar to studies conducted with other expansins in plants .

Why might I observe non-specific binding with my EXPA13 antibody?

Non-specific binding with EXPA13 antibodies can occur for several reasons, each requiring specific troubleshooting approaches:

• Cross-reactivity with other expansins:

  • Problem: High sequence homology between expansin family members

  • Solution: Pre-absorb antibody with recombinant proteins of closely related expansins (particularly EXPA1, EXPA3, EXPA8, EXPA9, EXPA10, and EXPA15)

  • Validation: Test antibody specificity on tissues from EXPA13 knockout plants

• Suboptimal blocking conditions:

  • Problem: Insufficient blocking allows antibody binding to non-specific sites

  • Solution: Increase blocking reagent concentration (5% BSA or milk), extend blocking time (2-3 hours), or try alternative blockers like casein or fish gelatin

  • Validation: Include control slides/blots with pre-immune serum

• Tissue fixation issues:

  • Problem: Overfixation can create artifactual binding sites

  • Solution: Optimize fixation time (2-4 hours maximum for paraformaldehyde), test milder fixatives

  • Validation: Compare different fixation protocols with the same antibody dilution

• Antibody concentration too high:

  • Problem: Excessive antibody increases background binding

  • Solution: Perform titration series (1:100 to 1:5000) to determine optimal concentration

  • Validation: Signal-to-noise ratio assessment at different dilutions

The careful validation approach used for OsEXPA10 antibody, where specificity was confirmed using genetic knockout lines, demonstrates an effective strategy to address these issues .

How should I troubleshoot inconsistent EXPA13 detection between experiments?

Inconsistent EXPA13 detection between experiments can be systematically addressed through the following troubleshooting framework:

• Sample preparation variability:

  • Problem: Inconsistent protein extraction efficiency

  • Solution: Standardize tissue collection (time of day, developmental stage), homogenization method, and buffer composition

  • Validation: Include internal loading controls (actin, tubulin) and total protein staining (Ponceau S)

• Antibody storage and handling:

  • Problem: Antibody degradation or aggregation

  • Solution: Aliquot antibodies to avoid freeze-thaw cycles, store with glycerol (50%) at -20°C

  • Validation: Include positive control sample across experiments

• Seasonal or environmental variation in plant material:

  • Problem: EXPA13 expression varies with growth conditions

  • Solution: Strictly control growth parameters (light, temperature, humidity) and document variations

  • Validation: Monitor expression of known EXPA13 regulators (e.g., MKK3, MPK7, ERF4)

• Technical variation in immunodetection:

  • Problem: Inconsistent transfer efficiency or detection sensitivity

  • Solution: Use stain-free gels or Ponceau S staining to confirm transfer, prepare fresh detection reagents

  • Validation: Include standard curve of recombinant protein

• Post-translational modifications:

  • Problem: Modifications affecting epitope recognition

  • Solution: Test multiple antibodies targeting different epitopes, use phosphatase treatment if phosphorylation is suspected

  • Validation: Mass spectrometry analysis to identify modifications

This systematic approach ensures reliable and reproducible detection of EXPA13 across different experimental conditions and plant materials.

What strategies can help distinguish between protein absence and antibody failure?

Distinguishing between true protein absence and antibody failure requires multiple validation approaches:

• Positive control inclusion:

  • Use recombinant EXPA13 protein at known concentrations

  • Include samples from EXPA13 overexpression lines

  • Test tissues known to express EXPA13 (e.g., imbibing seeds)

• Transcript analysis correlation:

  • Perform parallel RT-qPCR to determine EXPA13 mRNA levels

  • Compare protein detection results with transcript abundance

  • Consider time lag between transcription and translation

• Alternative antibody testing:

  • Use antibodies targeting different epitopes of EXPA13

  • Compare polyclonal vs. monoclonal antibody results

  • Try antibodies from different vendors or production batches

• Method validation:

  Validation Approach	Purpose	Expected Outcome
  Dot blot	Test antibody reactivity independent of gel separation	Signal with purified antigen
  Western following IP	Enrich target protein before detection	Enhanced signal from target
  Mass spectrometry	Direct protein identification	Peptide matches to EXPA13 sequence

• Protein extraction optimization:

  • Test multiple extraction buffers with different detergents and salt concentrations

  • Include protease inhibitors to prevent degradation

  • Optimize sample preparation to maintain protein integrity

These combined approaches can reliably determine whether negative results stem from actual protein absence or technical limitations with the antibody or detection method, similar to the comprehensive validation performed for OsEXPA10 antibodies .

How are EXPA13 antibodies being used to study stress responses in plants?

EXPA13 antibodies are increasingly being utilized to study plant stress responses, revealing important insights into adaptative mechanisms:

• Drought stress responses:

  • Immunolocalization studies show redistribution of EXPA13 in root tissues during water deficit

  • Western blot analyses reveal post-translational modifications of EXPA13 under drought conditions

  • Co-immunoprecipitation identifies stress-specific protein interaction partners

• Temperature stress adaptation:

  • EXPA13 abundance increases during cold acclimation in specific tissues

  • Heat stress induces rapid changes in EXPA13 distribution and abundance

  • Antibody-based chromatin studies reveal temperature-responsive transcription factors regulating EXPA13

• Oxidative stress signaling:

  • H₂O₂ treatment induces EXPA13 expression through the MKK3-MPK7 signaling pathway

  • Immunoprecipitation studies reveal oxidation-sensitive modifications of EXPA13

  • Co-localization with ROS sensors demonstrates spatial coordination of stress response

• Methodological innovations:

  • In situ proximity ligation assays with EXPA13 antibodies to detect protein-protein interactions in fixed tissues

  • Super-resolution microscopy to track EXPA13 localization changes during stress

  • Combination of EXPA13 immunodetection with metabolomic analyses to correlate protein levels with stress metabolites

These research applications demonstrate how EXPA13 antibodies can reveal the molecular mechanisms underlying plant adaptation to environmental stresses, particularly in seed germination under challenging conditions.

What emerging techniques are enhancing the specificity and sensitivity of EXPA13 detection?

Emerging techniques are significantly enhancing the specificity and sensitivity of EXPA13 detection in plant systems:

• Single-molecule detection approaches:

  • Single-molecule pull-down (SiMPull) combining antibody-based purification with single-molecule fluorescence

  • Total internal reflection fluorescence (TIRF) microscopy for surface-bound EXPA13 detection

  • Digital ELISA platforms providing femtomolar sensitivity

• Proximity-based detection methods:

  • Proximity ligation assay (PLA) for detecting EXPA13 interactions with cell wall components

  • APEX2-based proximity labeling to identify proteins in the EXPA13 microenvironment

  • Split-protein complementation assays to visualize EXPA13 interactions in living cells

• Antibody engineering advances:

  • Recombinant antibody fragments (Fab, scFv) with enhanced specificity for EXPA13

  • Nanobodies (VHH fragments) providing access to epitopes in confined cell wall spaces

  • Site-specific conjugation strategies for optimal reporter attachment

• Multiplexed detection systems:

  • Multiplexed immunofluorescence to simultaneously detect multiple expansins

  • Mass cytometry (CyTOF) with metal-labeled antibodies for high-parameter analysis

  • DNA-barcoded antibodies for spatial transcriptomics integration

• Direct tissue proteomics:

  • Imaging mass spectrometry with antibody-guided region identification

  • Digital spatial profiling combining immunofluorescence with spatially-resolved proteomics

  • Machine learning algorithms for automated signal quantification and pattern recognition

These technological advances address previous limitations in detecting low-abundance EXPA13 in complex plant tissues and enable more sophisticated studies of EXPA13 dynamics during plant development and stress responses.

How can EXPA13 antibodies contribute to understanding the evolutionary conservation of expansin function?

EXPA13 antibodies offer powerful tools for investigating evolutionary conservation of expansin function across plant species:

• Cross-species immunoreactivity studies:

  • Testing EXPA13 antibody recognition across diverse plant lineages (mosses, ferns, gymnosperms, angiosperms)

  • Mapping epitope conservation through comparative immunoblotting

  • Correlating antibody binding with functional conservation in cell wall loosening assays

• Developmental pattern comparisons:

  • Immunolocalization of EXPA13 homologs during key developmental stages across species

  • Comparison of tissue-specific expression patterns between monocots and dicots

  • Correlation of EXPA13 localization with specialized adaptations (e.g., seed dormancy mechanisms)

• Regulatory network conservation:

  • Using EXPA13 antibodies to study conservation of the MKK3-MPK7-ERF4 regulatory module across species

  • ChIP studies to compare transcription factor binding to EXPA13 promoters in diverse plants

  • Immunoprecipitation of regulatory complexes to identify conserved and divergent interactors

• Methodology for evolutionary studies:

  Approach	Application	Evolutionary Insight
  Epitope mapping	Identify conserved regions	Functional constraints on protein structure
  Western blot panel	Compare protein size/abundance	Diversification of expansin regulation
  Immunohistochemistry	Compare tissue distribution	Evolution of developmental patterning
  Co-IP in multiple species	Compare protein interactions	Conservation of molecular networks

• Integration with phylogenomics:

  • Correlation of antibody cross-reactivity with phylogenetic distance

  • Mapping of epitope conservation onto molecular evolution models

  • Identification of lineage-specific expansin adaptations

This evolutionary approach using EXPA13 antibodies provides insights into the fundamental mechanisms of plant cell growth that have been conserved or modified throughout plant evolution, particularly in seed plants where expansins play critical roles in germination.