EXPA3 Antibody

What is EXPA3 and what biological functions does it serve in Arabidopsis thaliana?

EXPA3 (Expansin A3) belongs to the α-expansin family of proteins that mediate acid-induced cell wall loosening in plants. In Arabidopsis thaliana, EXPA3 (UniProt accession: O80932) functions primarily in modifying cell wall extensibility during plant growth and development. The protein facilitates cell expansion by disrupting hydrogen bonds between cellulose microfibrils and cross-linking glycans in the cell wall matrix. EXPA3 expression is particularly notable during specific developmental stages, including seedling growth, root elongation, and leaf development. Understanding EXPA3's role is essential for broader research into plant growth mechanics, stress responses, and developmental biology .

What are the key specifications of commercially available EXPA3 Antibodies?

EXPA3 Antibodies designed for Arabidopsis thaliana research typically recognize specific epitopes of the EXPA3 protein (O80932). Commercial antibodies such as CSB-PA530516XA01DOA are available in various volume options (typically 0.1ml or 2ml) and are generally produced in rabbit hosts as polyclonal antibodies. These antibodies undergo purification processes (often using Protein A) to ensure specificity and reduced background signal. Most EXPA3 antibodies are designed for multiple applications including Western blotting, immunohistochemistry, and immunofluorescence techniques, enabling versatile experimental approaches in plant molecular biology research .

How should researchers validate the specificity of EXPA3 Antibody before experimental use?

Validating EXPA3 Antibody specificity is critical for reliable experimental outcomes. Researchers should first perform Western blot analysis using wild-type Arabidopsis protein extracts alongside expa3 mutant or knockdown lines as negative controls. A specific band at approximately 25-30 kDa (the expected molecular weight of EXPA3) should appear only in wild-type samples. Pre-absorption tests using the immunizing peptide can further confirm specificity - when the antibody is pre-incubated with excess antigen peptide, the signal should be substantially reduced or eliminated. Additionally, researchers should conduct immunohistochemistry on both wild-type and expa3 mutant tissues to validate tissue-specific staining patterns. Cross-reactivity with other expansin family members should be assessed through comparative analysis with recombinant expansin proteins, as the expansin family contains multiple members with structural similarities .

What sample preparation methods are recommended for optimal EXPA3 detection in plant tissues?

For optimal EXPA3 detection, tissue fixation and preservation methods must maintain both protein antigenicity and cellular architecture. Fresh Arabidopsis tissues should be fixed in 4% paraformaldehyde for 2-4 hours at room temperature, followed by gradual dehydration through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%). For paraffin embedding, tissues should be cleared with xylene before infiltration. For cryosectioning, tissues should be embedded in optimal cutting temperature (OCT) compound after fixation. Section thickness of 5-10 μm is recommended for most applications. For protein extraction in Western blotting, tissues should be ground in liquid nitrogen and homogenized in extraction buffer containing protease inhibitors, with particular attention to preventing proteolytic degradation during extraction. Cell wall-associated proteins like EXPA3 may require specialized extraction buffers with higher salt concentrations (typically 1M NaCl) to release proteins bound to cell wall components. Gentle agitation overnight at 4°C often improves extraction efficiency of cell wall-associated expansins .

How can differential expression of EXPA3 be accurately quantified across various developmental stages and stress conditions?

Accurate quantification of EXPA3 expression requires a multi-method approach combining transcriptomic and proteomic techniques. For transcript-level analysis, RT-qPCR should be performed using EXPA3-specific primers designed to span exon junctions to prevent genomic DNA amplification. Reference genes such as ACTIN2, UBQ10, or PP2A are recommended for normalization in Arabidopsis studies. For protein-level quantification, quantitative Western blotting using the EXPA3 antibody with chemiluminescent or fluorescent detection systems provides reliable results. For spatial expression patterns, researchers should generate EXPA3 promoter-reporter constructs (such as EXPA3pro:GUS or EXPA3pro:GFP) for visualization in transgenic plants. For stress response studies, a time-course experimental design is crucial, with sampling at multiple timepoints (0, 1, 3, 6, 12, 24, and 48 hours) after stress application. RNA-seq analysis can provide genome-wide context for EXPA3 expression changes, while proteomic approaches like MRM (Multiple Reaction Monitoring) mass spectrometry can precisely quantify EXPA3 protein levels. Cross-validation between transcript and protein levels is essential, as post-transcriptional regulation often affects expansin functionality .

What methodological approaches are most effective for investigating EXPA3 interactions with cell wall components?

Investigating EXPA3 interactions with cell wall components requires sophisticated biochemical and imaging techniques. Co-immunoprecipitation (Co-IP) using the EXPA3 antibody can identify protein-protein interactions, while pull-down assays with recombinant EXPA3 and purified cell wall fractions can reveal binding affinities to specific cell wall polymers. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) should be employed to determine binding kinetics and thermodynamic parameters of EXPA3 interactions with cellulose, hemicellulose, or pectin components. For in situ analysis, researchers should utilize proximity ligation assays (PLA) or Förster resonance energy transfer (FRET) with fluorescently-tagged EXPA3 and labeled cell wall components. Atomic force microscopy (AFM) with functionalized tips containing immobilized EXPA3 can map binding sites on isolated cell wall sections with nanometer resolution. Functional analysis through cell wall extensibility assays (creep tests) with purified cell walls and recombinant EXPA3 protein can establish direct mechanical effects on wall properties, while correlation with wall composition analysis (using techniques like FTIR spectroscopy or NMR) can reveal substrate preferences .

How can researchers effectively troubleshoot non-specific binding or weak signal problems when using EXPA3 Antibody in immunolocalization experiments?

When troubleshooting immunolocalization with EXPA3 antibody, researchers should first optimize blocking conditions by testing different blocking agents (BSA, normal serum, casein, or commercial blockers) at various concentrations (3-5%) and incubation times (1-3 hours). For high background issues, increasing washing steps (at least 3x15 minutes with 0.1-0.3% Tween-20 in PBS) and diluting primary antibody (typically 1:500 to 1:2000) can significantly improve signal-to-noise ratio. For weak signals, antigen retrieval methods should be tested, including heat-induced (citrate buffer, pH 6.0, at 95°C for 10-20 minutes) or enzymatic retrieval (proteinase K at 20 μg/ml for 10-15 minutes). Signal amplification systems such as tyramide signal amplification (TSA) or polymer-based detection systems can enhance sensitivity by 10-50 fold. When working with recalcitrant plant tissues, researchers should pre-treat sections with cell wall degrading enzymes (cellulase and pectinase mixture, 2% for 30 minutes at 37°C) to improve antibody penetration. Using alternative fixatives like zinc-based fixatives instead of aldehydes can sometimes preserve epitopes better. Technical controls must include omission of primary antibody, isotype controls, and validation in expa3 knockout lines. For quantitative analysis, researchers should employ standardized image acquisition parameters and analyze signal intensity using software like ImageJ with appropriate background subtraction .

What are the optimal experimental designs for investigating EXPA3's role in specific developmental processes and stress responses?

Investigating EXPA3's role in developmental processes requires comprehensive genetic, molecular, and physiological approaches. Researchers should generate and characterize multiple genetic resources: expa3 knockout mutants, EXPA3-overexpression lines, and inducible systems (such as estradiol-inducible or heat-shock inducible EXPA3 expression) for temporal control. For cell-type specific studies, develop transgenic lines expressing EXPA3 under tissue-specific promoters. Phenotypic analyses should include detailed growth measurements (primary root length, lateral root number, hypocotyl elongation, leaf expansion rate) using automated phenotyping platforms if available. For stress response studies, expose plants to precisely controlled stress conditions (drought with defined soil water potential, salt stress with carefully titrated NaCl concentrations, temperature stress with controlled ramping rates) and measure both EXPA3 expression changes and corresponding physiological parameters (osmotic adjustment, reactive oxygen species accumulation, membrane integrity). Live-cell imaging using EXPA3-fluorescent protein fusions can track protein localization dynamics during development or stress responses. Cell wall analysis techniques (comprehensive microarray polymer profiling, Fourier-transform infrared spectroscopy, atomic force microscopy) should be employed to correlate EXPA3 activity with specific wall structural changes. For systems-level understanding, integrate transcriptomic, proteomic, and metabolomic data from wild-type and expa3 mutant plants to construct gene regulatory networks and metabolic pathways influenced by EXPA3 activity .

What is the current understanding of post-translational modifications of EXPA3 and how can they be effectively studied?

EXPA3, like other expansins, undergoes several post-translational modifications (PTMs) that regulate its activity, localization, and stability. Glycosylation is particularly important for expansin function, with N-glycosylation sites potentially affecting protein folding and secretion through the endoplasmic reticulum. Phosphorylation may regulate EXPA3 activity in response to environmental signals or developmental cues. To study these PTMs, researchers should employ mass spectrometry-based approaches, including enrichment strategies for specific modifications. For glycosylation analysis, enzymatic deglycosylation (using PNGase F or Endo H) followed by mobility shift detection on Western blots using EXPA3 antibody can identify glycosylated forms. For comprehensive PTM mapping, purify EXPA3 using immunoprecipitation with the EXPA3 antibody followed by LC-MS/MS analysis. Phosphorylation studies should include phospho-specific antibody generation if phosphorylation sites are identified, or use of Phos-tag SDS-PAGE to separate phosphorylated from non-phosphorylated forms. Site-directed mutagenesis of identified PTM sites in EXPA3 followed by functional complementation assays in expa3 mutant plants can establish the biological significance of specific modifications. For in vivo dynamics, phospho-specific or glyco-specific staining methods can be combined with immunolocalization using the EXPA3 antibody. Integrating PTM data with structural modeling of EXPA3 can provide insights into how modifications affect protein conformation and function .

What is the recommended protocol for using EXPA3 Antibody in Western blot analysis of plant samples?

For Western blot analysis with EXPA3 Antibody, begin with optimized protein extraction. Grind 100-200 mg of Arabidopsis tissue in liquid nitrogen and homogenize in 500 μl extraction buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 1× protease inhibitor cocktail). For optimal extraction of cell wall-associated EXPA3, include an additional extraction step with high-salt buffer (1M NaCl in 50 mM Tris-HCl pH 8.0) for 2 hours at 4°C with gentle agitation. Centrifuge at 14,000×g for 15 minutes at 4°C and collect supernatant. Determine protein concentration using Bradford assay or BCA method. For gel electrophoresis, load 20-40 μg total protein per lane on a 12% SDS-PAGE gel, with appropriate molecular weight markers. Transfer proteins to PVDF membrane (recommended over nitrocellulose for plant samples) at 100V for 1 hour in cold transfer buffer. Block membrane with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature. Incubate with EXPA3 antibody at 1:1000 dilution in blocking buffer overnight at 4°C. Wash membrane 3×10 minutes with TBST. Incubate with HRP-conjugated secondary antibody (anti-rabbit IgG, 1:5000) for 1 hour at room temperature. Wash 3×10 minutes with TBST. Develop using enhanced chemiluminescence detection and expose to X-ray film or image using a digital imaging system. Expected band size for EXPA3 is approximately 25-30 kDa. For validation, include protein extracts from expa3 mutant plants as negative control .

How can researchers effectively use EXPA3 Antibody for immunolocalization in plant tissues?

For effective immunolocalization of EXPA3 in plant tissues, begin with proper fixation. Harvest fresh Arabidopsis tissues and immediately fix in 4% paraformaldehyde in PBS (pH 7.4) for 4 hours at room temperature under vacuum infiltration. After fixation, wash tissues 3×15 minutes in PBS. For paraffin embedding, dehydrate tissues through an ethanol series (30%, 50%, 70%, 95%, 100%, 30 minutes each), clear with xylene (2×30 minutes), and infiltrate with paraffin at 60°C (3 changes, 1 hour each). Section embedded tissues at 8 μm thickness using a microtome and mount on adhesive slides. For immunostaining, deparaffinize sections in xylene (2×10 minutes) and rehydrate through descending ethanol series to water. Perform antigen retrieval by heating slides in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 10 minutes, then cool to room temperature. Block endogenous peroxidase activity with 3% H₂O₂ in methanol for 10 minutes if using HRP-based detection. Block non-specific binding with 3% BSA in PBS containing 0.1% Triton X-100 for 1 hour at room temperature. Incubate with EXPA3 antibody diluted 1:200 in blocking solution overnight at 4°C in a humid chamber. Wash slides 3×10 minutes in PBS with 0.1% Tween-20. Incubate with appropriate secondary antibody (fluorescent-conjugated or HRP-conjugated anti-rabbit IgG, 1:500) for 1 hour at room temperature. For fluorescent detection, wash 3×10 minutes in PBS with 0.1% Tween-20 and counterstain nuclei with DAPI (1 μg/ml) for 5 minutes. Mount slides with anti-fade mounting medium. For enzymatic detection, incubate with DAB substrate until color develops (typically 5-10 minutes), then counterstain with hematoxylin. Mount slides with permanent mounting medium. Include negative controls (primary antibody omission, pre-immune serum, expa3 mutant tissues) in each experiment .

What techniques can be used to compare EXPA3 with other expansin family members in functional studies?

To compare EXPA3 with other expansin family members functionally, researchers should employ a systematic approach combining molecular, biochemical, and physiological techniques. Begin with sequence and structural analysis of EXPA3 alongside other expansins using bioinformatics tools to identify conserved domains and unique features. Generate multiple transgenic lines: (1) single knockout mutants for each expansin of interest, (2) multiple knockout combinations including expa3, (3) complementation lines expressing each expansin under the EXPA3 promoter in the expa3 background, and (4) chimeric proteins swapping domains between EXPA3 and other expansins. For protein activity assays, express and purify recombinant expansins in heterologous systems (preferably Pichia pastoris for proper post-translational modifications) and conduct in vitro extensibility assays using a constant-load extensometer with various cell wall substrates. Compare kinetic parameters (optimal pH, temperature, substrate specificity) systematically. For gene expression patterns, perform highly detailed expression profiling using a combination of qRT-PCR, promoter-reporter fusions, and in situ hybridization across multiple tissues, developmental stages, and stress conditions to identify overlapping and distinct expression domains. For protein-protein interactions, conduct yeast two-hybrid or bimolecular fluorescence complementation (BiFC) assays to identify differential interaction partners. Cell wall analysis should include comprehensive microarray polymer profiling (CoMPP) and Fourier-transform infrared spectroscopy (FTIR) to correlate expansin activity with specific cell wall modifications. For functional redundancy assessment, analyze phenotypes of single and higher-order mutants under various growth conditions and stresses, focusing on parameters like cell expansion rates, organ growth kinetics, and biomechanical properties .

What statistical approaches are recommended for analyzing EXPA3 localization patterns across different tissue types and experimental conditions?

For robust statistical analysis of EXPA3 localization patterns, researchers should implement multi-level quantitative approaches. Begin with image acquisition standardization - capture at least 10-15 independent fields per tissue type using identical microscope settings (exposure time, gain, laser power) with appropriate technical replicates. For signal quantification, use specialized software (ImageJ with appropriate plugins, CellProfiler, or Imaris) to measure parameters including signal intensity, signal distribution patterns, colocalization coefficients with subcellular markers, and proportion of cells showing specific localization patterns. Implement proper thresholding methods (preferably automated or observer-blinded) to distinguish signal from background. For statistical testing, use nested ANOVA or linear mixed models to account for biological variation (different plants, different tissues) and technical variation (slide-to-slide, field-to-field). For complex localization patterns, employ multivariate statistical approaches such as principal component analysis or discriminant analysis to identify key variables distinguishing experimental conditions. For colocalization studies, calculate Pearson's or Mander's coefficients and test significance using appropriate randomization tests. For comparing proportions of cells with specific localization patterns, use chi-square tests or logistic regression. For time-series data, consider repeated measures ANOVA or longitudinal data analysis techniques. Report effect sizes alongside p-values to indicate biological significance. For comprehensive documentation, present both representative images and quantitative data visualization (box plots, violin plots) with appropriate statistical annotations. Minimum biological replicates should include 3-5 independent experiments with different plant batches .

What are the emerging research directions for EXPA3 Antibody applications in plant developmental biology?

Emerging research directions for EXPA3 Antibody applications are expanding beyond traditional protein detection toward more sophisticated methodologies that integrate multiple disciplines. Super-resolution microscopy techniques including STORM, PALM, and STED microscopy are increasingly being applied with EXPA3 antibodies to visualize expansin distribution within cell walls at nanometer resolution, revealing previously undetectable spatial patterns and protein clustering. Live-cell imaging applications using membrane-permeable EXPA3 antibody fragments or nanobodies are emerging to track dynamic changes in EXPA3 localization during rapid growth responses. Multi-epitope imaging approaches combining EXPA3 antibodies with probes for specific cell wall polymers enable simultaneous visualization of enzyme-substrate relationships. In synthetic biology applications, researchers are developing EXPA3-based biosensors by coupling antibody-based detection with reporter systems to monitor expansin activity in real-time. CRISPR-based genome editing combined with EXPA3 immunolocalization is revealing precise structure-function relationships by creating targeted modifications to specific EXPA3 domains. Single-cell approaches including single-cell proteomics with EXPA3 antibody-based detection are uncovering cell-to-cell variability in expansin expression that may explain differential growth patterns within tissues. These emerging approaches are particularly valuable for investigating EXPA3's role in sophisticated developmental processes including mechanosensing, tropisms, and cell fate determination during plant morphogenesis .