EXPA18 Antibody

What is EXPA18 and why is it significant in plant biology research?

EXPA18 (Expansin A18) is a member of the α-expansin family in rice (Oryza sativa subsp. japonica). Expansins are critical proteins that mediate cell wall loosening during plant growth, cell expansion, and developmental processes. EXPA18 specifically plays roles in rice development and stress responses. Studying EXPA18 provides insights into fundamental mechanisms of plant cell expansion, growth regulation, and environmental adaptation. The polyclonal antibody against EXPA18 enables researchers to detect, localize, and quantify this protein in various experimental contexts, facilitating studies on gene expression, protein localization, and functional characterization .

What are the validated applications for EXPA18 antibody?

The EXPA18 antibody has been specifically validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications in rice (Oryza sativa) samples. These techniques allow for both quantitative analysis and molecular weight determination of the target protein. While these are the confirmed applications, researchers should note that optimization may be required when applying this antibody to other techniques such as immunohistochemistry (IHC), immunocytochemistry (ICC), or immunoprecipitation (IP). Preliminary validation experiments are recommended when expanding to these additional applications .

How should EXPA18 antibody be stored to maintain optimal activity?

EXPA18 antibody should be stored at -20°C or ideally -80°C for long-term preservation of activity. Upon receipt, the antibody should be aliquoted to avoid repeated freeze-thaw cycles which can significantly degrade antibody performance. The formulation contains 50% glycerol in PBS (pH 7.4) with 0.03% Proclin 300 as a preservative, which helps maintain stability during storage. Each aliquot should contain sufficient antibody for a single experiment to minimize freeze-thaw cycles. When handling the antibody, always keep it on ice and return to freezer storage promptly after use .

What are the optimal sample preparation methods for Western blotting with EXPA18 antibody?

For effective Western blot analysis of EXPA18 in rice samples, follow this optimized protocol:

• Tissue extraction: Harvest fresh rice tissue and immediately flash-freeze in liquid nitrogen. Grind tissue to fine powder while maintaining frozen state.

• Protein extraction buffer: Use a plant-specific extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

  • 10 mM DTT (to reduce disulfide bonds)

  • 1 mM PMSF

• Extraction process: Add 5 ml buffer per gram of tissue powder. Vortex and incubate on ice for 30 minutes with intermittent mixing.

• Clarification: Centrifuge at 15,000 × g for 20 minutes at 4°C. Collect supernatant.

• Sample preparation: Mix protein extract with Laemmli buffer (containing SDS and β-mercaptoethanol). Heat at 95°C for 5 minutes.

• Gel selection: Use 10-12% polyacrylamide gels for optimal separation of EXPA18 (~25-30 kDa range).

• Blocking: Block membrane with 5% non-fat dry milk or 5% BSA in TBST for 1 hour.

• Antibody dilution: Dilute EXPA18 antibody 1:1000 in blocking buffer. Incubate overnight at 4°C.

• Controls: Include both positive control (rice tissue known to express EXPA18) and negative control (non-plant tissue or pre-immune serum) .

This protocol enhances specificity and reduces background by accounting for the unique challenges of plant tissue protein extraction, including high levels of polysaccharides and phenolic compounds.

How can I optimize ELISA protocols for quantitative analysis of EXPA18?

To optimize ELISA for EXPA18 quantification in plant samples, implement this methodological approach:

Sandwich ELISA Protocol Optimization:

• Plate coating: Coat high-binding ELISA plates with capture antibody (1-10 μg/ml in carbonate buffer, pH 9.6) overnight at 4°C.

• Sample preparation: For plant tissues, use a specialized extraction buffer:

  • 100 mM Tris-HCl (pH 8.0)

  • 150 mM NaCl

  • 5 mM EDTA

  • 0.1% Tween-20

  • 1 mM PMSF

  • Plant protease inhibitor cocktail

• Dilution series: Create a standard curve using recombinant EXPA18 protein (0.1-1000 ng/ml) to ensure quantitative accuracy.

• Detection approach: Implement a two-antibody system similar to successful cytokine detection systems, where different antibodies recognize distinct epitopes of EXPA18 .

• Signal enhancement: Consider using amplification systems (e.g., biotin-streptavidin) to improve sensitivity when detecting low abundance EXPA18.

• Cross-validation: Verify ELISA results with Western blot analysis to confirm specificity of detection.

This optimization approach addresses the unique challenges of plant tissue processing while providing quantitative reliability comparable to established mammalian systems .

What strategies should be employed to reduce non-specific binding when using EXPA18 antibody?

To minimize non-specific binding when working with EXPA18 antibody in plant samples, implement these research-validated strategies:

• Optimized blocking: Use 5% BSA rather than milk for blocking in Western blots when working with plant samples. Plant proteins may cross-react with milk proteins.

• Pre-absorption protocol:

  • Incubate diluted antibody with non-target plant tissue lysate (e.g., Arabidopsis if working with rice) at a ratio of 1:10 for 2 hours at room temperature

  • Centrifuge at 12,000 × g for 10 minutes to remove antibody-antigen complexes

  • Use the supernatant for immunodetection

• Buffer optimization: Add 0.1-0.3% Tween-20 and 0.1-0.5 M NaCl to wash buffers to reduce electrostatic interactions causing non-specific binding.

• Antigen-specific controls: Include competitive inhibition controls by pre-incubating antibody with recombinant EXPA18 protein prior to immunodetection.

• Cross-species validation: When possible, compare binding patterns across multiple plant species to identify conserved versus non-specific signals .

These approaches significantly improve signal-to-noise ratio based on principles established for antibody specificity optimization across diverse experimental systems.

How can computational modeling be used to predict EXPA18 antibody epitopes and improve experimental design?

Computational epitope prediction can significantly enhance experimental design when working with EXPA18 antibody:

• Sequence-based epitope prediction:

  • Utilize algorithms such as BepiPred, ABCpred, and SVMTriP to analyze the EXPA18 protein sequence (UniProt: Q4PR48)

  • Focus on regions with high surface accessibility and hydrophilicity

  • Compare predictions across multiple algorithms to identify consensus epitopes

• Structural considerations:

  • Generate 3D homology models of EXPA18 using AlphaFold or similar tools

  • Map predicted epitopes onto the structural model to verify surface exposure

  • Account for post-translational modifications that might affect epitope accessibility

• Experimental validation strategy:

  • Design peptide arrays covering predicted epitope regions

  • Test antibody binding to these peptides to confirm actual epitopes

  • Use this information to develop blocking peptides for specificity controls

• Cross-reactivity prediction:

  • Compare EXPA18 epitopes with other expansin family members to anticipate potential cross-reactivity

  • Design experiments with appropriate controls based on predicted cross-reactivity

The biophysics-informed modeling approach, similar to that described for antibody specificity inference, can significantly improve experimental outcomes by providing a rational basis for selecting epitopes and designing proper controls .

What are the considerations for using EXPA18 antibody in immunolocalization studies in plant tissues?

For successful immunolocalization of EXPA18 in plant tissues, researchers should address these critical considerations:

• Fixation optimization:

  • Test multiple fixatives: 4% paraformaldehyde preserves antigenicity but 2% glutaraldehyde may better preserve cell wall structures

  • Fixation time should be optimized (4-24 hours) depending on tissue thickness

  • Consider ethanol-acetic acid fixation (3:1) as an alternative for preserving cell wall proteins

• Tissue sectioning strategies:

  • For paraffin embedding: Use plant-optimized infiltration schedules with extended ethanol series

  • For cryosectioning: Utilize 1.5 M sucrose as cryoprotectant specific for plant tissues

  • Section thickness: 5-8 μm for light microscopy, 60-80 nm for electron microscopy

• Antigen retrieval techniques:

  • Enzymatic treatment: 1% cellulase + 1% pectinase for 15-30 minutes at room temperature

  • Heat-mediated: Citrate buffer (pH 6.0) at 95°C for 10-15 minutes

  • Both methods may be required sequentially for optimal EXPA18 detection

• Antibody penetration enhancement:

  • Include 0.1% Triton X-100 in washing and antibody dilution buffers

  • Extend primary antibody incubation to 48-72 hours at 4°C for thick sections

  • Consider vacuum infiltration for improved antibody penetration

• Controls specific for plant cell walls:

  • Pre-immune serum controls

  • Absorption controls with recombinant EXPA18

  • Secondary antibody-only controls

  • Comparative localization with other known cell wall proteins

These methodological refinements address the unique challenges of plant cell wall immunolocalization, including cell wall porosity limitations and autofluorescence issues .

How can EXPA18 antibody be used in studying plant stress responses?

EXPA18 antibody can be instrumental in elucidating plant stress response mechanisms through these research approaches:

• Temporal expression profiling:

  • Monitor EXPA18 protein levels at defined time points after stress application (0, 1, 3, 6, 12, 24, 48 hours)

  • Compare protein levels (Western blot) with transcript levels (qRT-PCR) to identify post-transcriptional regulation

  • Create expression kinetics curves to determine the timing of EXPA18 involvement in stress responses

• Spatial expression analysis:

  • Use immunohistochemistry to map EXPA18 distribution in different tissues under stress conditions

  • Compare control vs. stressed plants to identify stress-specific relocalization

  • Combine with in situ hybridization to distinguish between translocation and de novo synthesis

• Protein-protein interaction studies:

  • Employ co-immunoprecipitation with EXPA18 antibody followed by mass spectrometry

  • Identify stress-specific interaction partners that may regulate EXPA18 function

  • Validate interactions using techniques such as BiFC or FRET

• Cell wall modification analysis:

  • Correlate EXPA18 levels with cell wall extensibility measurements

  • Combine immunolocalization with cell wall component staining (calcofluor for cellulose, ruthenium red for pectins)

  • Assess how stress alters the relationship between EXPA18 localization and cell wall properties

This multifaceted approach leverages the EXPA18 antibody to connect molecular events with physiological responses during plant stress adaptation .

What are common issues when using EXPA18 antibody and how can they be resolved?

These troubleshooting strategies are adapted from both general antibody principles and specific considerations for plant cell wall proteins, addressing the unique challenges of plant tissue processing .

How can researchers validate the specificity of EXPA18 antibody in their experimental system?

To rigorously validate EXPA18 antibody specificity in plant experimental systems, implement this comprehensive validation protocol:

• Multiple detection techniques correlation:

  • Compare Western blot, ELISA, and immunohistochemistry results

  • Consistent molecular weight and localization patterns across methods suggest specificity

  • Discrepancies may indicate technique-specific artifacts

• Genetic validation:

  • Test antibody against EXPA18 knockout/knockdown lines

  • Use CRISPR-edited plants with epitope mutations

  • Overexpression lines should show increased signal intensity

• Peptide competition assay:

  • Pre-incubate antibody with purified recombinant EXPA18 protein (10-100-fold molar excess)

  • Specific signals should be significantly reduced or eliminated

  • Non-specific signals will remain unaffected

• Cross-species validation:

  • Test reactivity against homologous proteins from related plant species

  • Signal strength should correlate with sequence conservation of the epitope region

  • Create a phylogenetic specificity profile based on these results

• Mass spectrometry validation:

  • Perform immunoprecipitation with EXPA18 antibody

  • Analyze pulled-down proteins by LC-MS/MS

  • Confirm that EXPA18 peptides are identified in the bound fraction

• Orthogonal detection methods:

  • Compare antibody detection with GFP-tagged EXPA18 localization

  • Correlate with in situ hybridization of EXPA18 mRNA

  • Verify expression patterns match known transcriptomic data

This comprehensive validation approach, incorporating genetic, biochemical, and biophysical methods, ensures reliable experimental outcomes when working with EXPA18 antibody .

How should researchers interpret differences in EXPA18 protein levels versus transcript levels in their studies?

Discrepancies between EXPA18 protein and transcript levels are common in plant research and require careful interpretation:

• Temporal considerations:

  • Protein synthesis typically lags behind transcriptional changes (4-8 hour delay in plants)

  • Create time-course experiments capturing both transcript (qRT-PCR) and protein (Western blot) levels

  • Analyze the temporal relationship to distinguish between delayed translation versus post-transcriptional regulation

• Post-transcriptional regulation mechanisms:

  • Assess mRNA stability through actinomycin D treatment (transcription inhibitor)

  • Investigate miRNA involvement by correlating with expression of predicted miRNA regulators

  • Examine alternative splicing patterns through RT-PCR with exon-spanning primers

• Post-translational regulation factors:

  • Measure protein half-life using cycloheximide chase assays

  • Investigate ubiquitination status through immunoprecipitation with ubiquitin antibodies

  • Assess phosphorylation state using phospho-specific antibodies or phosphatase treatments

• Tissue-specific considerations:

  • Protein may be synthesized in one tissue and transported to another

  • Compare tissue-specific transcript levels with protein distribution

  • Use tissue-specific promoter reporters to track expression patterns

• Quantitative analytical approaches:

  • Calculate protein-to-mRNA ratios across conditions

  • Apply mathematical modeling to determine synthesis and degradation rates

  • Use these models to identify regulatory transition points

This interpretative framework helps researchers distinguish between transcriptional, post-transcriptional, and post-translational regulatory mechanisms affecting EXPA18 expression during plant development and stress responses .

How can EXPA18 antibody be adapted for high-throughput screening applications?

Adapting EXPA18 antibody for high-throughput screening requires systematic optimization of several parameters:

• Automated ELISA development:

  • Miniaturization to 384-well format requires reducing volumes by 60-75%

  • Optimize antibody concentration through checkerboard titration (1:500-1:10,000)

  • Implement robotic liquid handling systems for consistent delivery

  • Develop Z'-factor analysis for assay quality assessment (aim for Z' > 0.5)

• Multiplex detection systems:

  • Conjugate EXPA18 antibody with fluorescent dyes compatible with multiplex analysis

  • Validate spectral separation when combined with antibodies against other cell wall proteins

  • Optimize signal-to-noise ratios for each detection channel

  • Include automated image analysis algorithms for quantification

• Cell-based screening applications:

  • Adapt for high-content screening in plant protoplast systems

  • Develop image analysis pipelines specific for plant cell morphology

  • Incorporate machine learning algorithms for pattern recognition

  • Validate reproducibility across biological replicates (CV < 15%)

• Microarray-based approaches:

  • Spot recombinant proteins or plant extracts on nitrocellulose-coated slides

  • Optimize EXPA18 antibody concentration and incubation conditions for microarray format

  • Implement internal calibration standards for quantitative analysis

  • Develop normalized signal intensity metrics for cross-sample comparison

These adaptations enable screening of large sample sets, such as germplasm collections or stress treatment time courses, while maintaining quantitative precision and reproducibility .

What are the considerations for using EXPA18 antibody in combination with other antibodies for co-localization studies?

When designing co-localization studies with EXPA18 antibody and other antibodies, researchers should address these critical factors:

• Antibody compatibility assessment:

  • Primary antibody host species must differ (e.g., rabbit anti-EXPA18 with mouse anti-cellulose synthase)

  • If using multiple rabbit antibodies, employ sequential immunostaining with antibody elution steps

  • Validate that antibody elution does not affect tissue morphology or antigen preservation

• Fixation protocol harmonization:

  • Different proteins may require different optimal fixation methods

  • Test multiple fixation protocols to find one that preserves all antigens of interest

  • Consider dual fixation approaches (e.g., brief glutaraldehyde followed by paraformaldehyde)

• Signal discrimination optimization:

  • Select fluorophores with minimal spectral overlap (e.g., Alexa 488 and Alexa 647)

  • Include single-antibody controls to assess bleed-through

  • Apply spectral unmixing algorithms for closely overlapping fluorophores

  • Consider quantum dots for improved photostability in extended imaging sessions

• Antigen retrieval compatibility:

  • Different epitopes may require different retrieval methods

  • Test sequential application of retrieval methods

  • Optimize pH and temperature conditions for maximum compatibility

• Specialized controls for co-localization:

  • Include antibody omission controls for each primary antibody

  • Use biological samples with known expression patterns as positive controls

  • Apply quantitative co-localization analysis (Pearson's or Manders' coefficients)

  • Implement randomization tests to validate statistical significance of co-localization

These methodological considerations ensure reliable multi-protein localization analysis, particularly challenging in plant cell wall contexts where protein accessibility is often limited by cell wall structure .

How can researchers design custom experiments to study EXPA18 protein-protein interactions in planta?

To investigate EXPA18 protein-protein interactions in plant systems, implement these specialized experimental approaches:

• Optimized co-immunoprecipitation (Co-IP) protocol:

  • Use chemically crosslinkable detergents (e.g., DSP-containing lysis buffers) to stabilize transient interactions

  • Implement a two-step extraction: low-stringency initial extraction followed by more aggressive extraction

  • Add 0.5-1% cellulase to extraction buffer to partially digest cell walls and release wall-associated complexes

  • Validate with known interacting proteins (e.g., other expansins, cell wall modifying enzymes)

  • Control for non-specific interactions with pre-immune serum pulldowns

• Proximity-dependent labeling approaches:

  • Generate transgenic plants expressing EXPA18-BioID or EXPA18-TurboID fusions

  • Optimize biotin infiltration methods specific for plant tissues

  • Implement vacuum infiltration (5-10 min at 100 mbar) to ensure biotin penetration

  • Verify fusion protein functionality through complementation assays

  • Identify biotinylated proteins using mass spectrometry

• Förster Resonance Energy Transfer (FRET) analysis:

  • Create fluorescent protein fusions that preserve EXPA18 localization and function

  • Test multiple fusion orientations (N vs C terminal) to identify optimal configuration

  • Implement acceptor photobleaching FRET for plant cell wall localized proteins

  • Quantify FRET efficiency using standardized calculation methods

  • Compare results in different cell types and developmental stages

• Split-reporter protein complementation assays:

  • Develop EXPA18 fusions with split fluorescent proteins (e.g., split-YFP) or split luciferase

  • Screen against libraries of cell wall proteins to identify novel interactors

  • Validate interactions using targeted constructs and appropriate controls

  • Compare interaction profiles under normal versus stress conditions

  • Correlate with phenotypic changes in cell wall properties

These approaches, adapted specifically for plant cell wall proteins, enable comprehensive characterization of EXPA18's interactome and functional relationships in developmentally and environmentally relevant contexts .