EMB1144 Antibody

What is EMB1144 and why is it significant in plant research?

EMB1144 (embryo defective 1144) is a gene that encodes chorismate synthase, a critical enzyme involved in the shikimate pathway essential for aromatic amino acid biosynthesis in plants. The significance of EMB1144 stems from its fundamental role in embryo development, where mutations in this gene result in embryo-lethal phenotypes. Research using EMB1144 antibodies allows scientists to track the expression and localization of this protein during developmental processes and under various environmental conditions. The protein is particularly important because it represents a class of proteins essential for early plant development, sharing characteristics with other EMB proteins identified through forward genetic screens designed to isolate embryo-defective mutants .

How does EMB1144 relate to embryo development in plants?

EMB1144 is crucial for proper embryo development in plants, particularly in Arabidopsis thaliana. The gene was identified through forward genetic screens specifically designed to isolate embryo-defective (emb) mutants. Mutations in EMB1144 lead to arrested embryo development, resulting in seed abortion. This phenotype indicates that the protein functions in essential metabolic pathways required for embryogenesis.

Similar to other identified embryo-defective genes like BIO1, EMB1144 mutants show characteristic developmental arrest at specific embryonic stages. The embryo-lethal phenotype can sometimes be rescued through nutrient supplementation on enriched media, suggesting its role in vital biosynthetic pathways . The protein's involvement in chorismate synthesis connects it to the production of aromatic amino acids and various secondary metabolites essential for embryo maturation and development.

What techniques can be used to detect EMB1144 protein in plant tissues?

For effective detection of EMB1144 protein in plant tissues, researchers can employ several complementary techniques:

• Western Blotting: Protein extracts from plant tissues can be separated using SDS-PAGE (12.5% gels recommended) and transferred to membranes for immunodetection with anti-EMB1144 antibodies. For optimal results, use a primary antibody dilution of 1:1000, followed by an appropriate HRP-conjugated secondary antibody (goat anti-rabbit at 1:2500 dilution). Visualization can be achieved using standard ECL substrates .

• Immunohistochemistry: For tissue-specific localization, plant samples should be fixed, embedded, sectioned, and subjected to antigen retrieval before antibody incubation. This allows visualization of the spatial distribution of EMB1144 across different cell types.

• Immunoprecipitation: EMB1144 antibodies can be used to isolate the protein and its interacting partners from plant extracts, enabling studies of protein complexes and post-translational modifications.

• Fluorescence Microscopy: Combining EMB1144 antibodies with fluorescently-labeled secondary antibodies allows subcellular localization studies, particularly valuable when examining expression in specific cell types like those in the root cortex versus the stele .

How is EMB1144 expression affected by abiotic stresses?

EMB1144 expression exhibits significant changes in response to various abiotic stresses, particularly salinity. Based on transcriptomic analyses:

Salt Stress Response:

• In cortical cells, EMB1144 expression shows notable regulation as part of the plant's adaptation to salinity .

• The gene's response differs between cell types, with distinct patterns observed in cortical versus pericycle cells.

Other Abiotic Stresses:

• High light, heat stress, and drought conditions can also influence EMB1144 expression patterns, similar to other stress-responsive genes involved in plant metabolism .

• Under drought conditions, EMB1144 expression changes correlate with alterations in advanced glycation end products (AGEs), suggesting a potential connection between this protein and stress-induced post-translational modifications .

The cell type-specific nature of these responses highlights the importance of tissue-specific analysis when studying EMB1144 expression under stress conditions. Transcriptomic data reveals that EMB1144 functions within broader networks of genes responding to environmental challenges, particularly those affecting plant water relations and cell wall composition .

What are the optimal sample preparation methods for EMB1144 antibody-based experiments?

For optimal results with EMB1144 antibody-based experiments, sample preparation should be tailored to specific experimental applications:

For Protein Extraction and Western Blotting:

• Harvest fresh plant tissue and flash-freeze in liquid nitrogen.

• Grind tissue to a fine powder while maintaining freezing temperatures.

• Extract proteins using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, and protease inhibitor cocktail.

• Quantify protein concentration using the Bradford assay.

• For SDS-PAGE, load 10-20μg of total protein per lane on a 12.5% gel.

• For immunoblotting, use a 1:1000 dilution of the primary EMB1144 antibody followed by a 1:2500 dilution of HRP-conjugated secondary antibody .

For Immunoprecipitation:

• Extract proteins in a milder buffer (25mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40) to preserve protein-protein interactions.

• Pre-clear lysates with Protein A/G beads.

• Incubate with EMB1144 antibody (5μg per 1mg of total protein) overnight at 4°C.

• Add Protein A/G beads and incubate for 2-3 hours.

• Wash thoroughly and elute for downstream analysis .

For Immunohistochemistry:

• Fix tissue samples in 4% paraformaldehyde for 12-16 hours.

• Dehydrate through an ethanol series and embed in paraffin or resin.

• Section tissues at 5-10μm thickness.

• Perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 20 minutes.

• Block with 3% BSA in PBS for 1 hour and incubate with EMB1144 antibody at 1:100 dilution overnight at 4°C .

These optimized protocols will help ensure specific detection of EMB1144 while minimizing background and false positives in different experimental contexts.

How can EMB1144 antibodies be validated for specificity?

Validating the specificity of EMB1144 antibodies is crucial for reliable research results. A comprehensive validation approach should include:

Genetic Controls:

• Knockout/Knockdown Verification: Test the antibody on tissues from EMB1144 knockout or knockdown lines. Since complete knockouts may be embryo-lethal, use inducible or tissue-specific knockdown approaches. The absence or significant reduction of signal in these samples compared to wild-type confirms specificity .

• Overexpression Analysis: Test antibodies on samples overexpressing EMB1144 to confirm increased signal intensity correlating with expression levels.

Biochemical Validation:

• Western Blot Analysis: Verify that the antibody detects a single band of the expected molecular weight (~55-60 kDa for chorismate synthase). Multiple bands might indicate cross-reactivity or post-translational modifications .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide before application to samples. Signal disappearance indicates specific binding to the target epitope.

• Mass Spectrometry Confirmation: Immunoprecipitate the protein using the antibody and confirm its identity through MS/MS analysis, comparing results with known EMB1144 peptide sequences .

Technical Controls:

• Secondary Antibody-Only Control: Omit primary antibody to assess background from secondary antibody binding.

• Cross-Species Reactivity: Test antibody against EMB1144 homologs in related plant species to determine conservation of the epitope recognition .

Additional Validation Methods:

• Immunohistochemistry Patterns: Verify that localization patterns match expected tissue and subcellular distribution based on transcriptomic data.

• Reciprocal Isolation: Perform reciprocal co-immunoprecipitation experiments to confirm specific protein-protein interactions .

Comprehensive validation using multiple approaches ensures reliable results and minimizes false positives in EMB1144 antibody-based research.

What protein-protein interactions have been documented for EMB1144?

EMB1144, as a chorismate synthase involved in critical metabolic pathways, participates in several protein-protein interactions that are essential for its function and regulation. While direct interaction data for EMB1144 specifically is limited, several important interactions can be inferred based on studies of chorismate synthase and related proteins in plants:

Metabolic Enzyme Complexes:

• EMB1144 likely forms functional complexes with other enzymes in the shikimate pathway, enabling metabolic channeling and enhanced efficiency of aromatic amino acid biosynthesis.

• Evidence suggests interactions with DAHP synthase and shikimate kinase, creating a multi-enzyme complex that facilitates substrate transfer between consecutive reactions.

Regulatory Interactions:

• Under stress conditions, EMB1144 may interact with regulatory proteins involved in redox homeostasis, similar to the documented interactions between stress-responsive proteins and antioxidant defense mechanisms .

• Potential interaction with thioredoxins (TRX3 and TRX5) as part of redox-dependent regulation systems, particularly under oxidative stress conditions .

Detection Methods:
For studying EMB1144 protein interactions, researchers commonly employ:

• Co-immunoprecipitation (Co-IP): Using EMB1144 antibodies to pull down the protein along with its interacting partners from plant extracts.

• Tandem Affinity Purification coupled with Mass Spectrometry (TAP-MS): A two-step purification approach that reduces false positives when identifying protein complexes .

• Yeast Two-Hybrid Screening: To detect direct binary interactions between EMB1144 and potential partners.

Validation Approaches:

• Reciprocal isolation methods where both EMB1144 and its suspected interaction partners are used as bait in separate experiments .

• Confirmation of co-localization using fluorescently tagged proteins or dual immunostaining approaches.

Further research using techniques such as proximity-dependent biotin identification (BioID) or crosslinking mass spectrometry could reveal additional interactions and provide deeper insights into EMB1144's functional networks in plant cells.

How can EMB1144 antibodies be used in cell type-specific studies?

EMB1144 antibodies offer powerful tools for investigating cell type-specific expression and function in plant tissues. Implementation of these approaches requires careful consideration of tissue preparation, antibody application, and analysis methods:

Cell Type Isolation Methods:

• Fluorescence-Activated Cell Sorting (FACS): When combined with cell type-specific fluorescent markers (e.g., GFP enhancer trap lines), tissues can be dissociated and sorted for subsequent EMB1144 immunodetection. This approach has been successfully used to isolate specific cell types from root tissues for transcriptomic analysis .

• Laser Capture Microdissection: Allows collection of specific cell types from fixed tissue sections for subsequent protein extraction and EMB1144 antibody-based detection.

Immunohistochemical Applications:

• Double Immunolabeling: Combine EMB1144 antibodies with markers for specific cell types (e.g., cortical cells vs. stelar tissues) to determine relative expression levels across different root domains .

• In situ Western Analysis: Perform protein blotting directly on tissue sections to maintain spatial information while detecting EMB1144 protein.

Transcriptomics Integration:

• Compare EMB1144 protein distribution detected by antibodies with transcriptomic data from cell type-specific studies. For instance, studies have revealed distinctive gene expression patterns between cortical and stelar tissues in roots, which can be correlated with protein localization .

• Integrate antibody detection with data from enhancer trap lines expressing GFP in specific cell types, such as those used in Arabidopsis and rice root studies .

Experimental Design Considerations:

• Use GAL4 enhancer trap lines that mark specific cell types (e.g., pericycle vs. cortical cells) to correlate EMB1144 protein detection with known cell identity markers .

• When studying stress responses, consider the differential regulation observed between cell types. For example, cortical and pericycle cells show distinct transcriptional responses to salt stress that might be reflected in EMB1144 protein levels and modifications .

This cell type-specific approach reveals functional heterogeneity that would be missed in whole-tissue analyses, providing deeper insights into EMB1144's role in plant development and stress responses.

What are the technical challenges in detecting post-translational modifications of EMB1144?

Detecting post-translational modifications (PTMs) of EMB1144 presents several technical challenges that require sophisticated approaches to overcome:

Challenge 1: Low Abundance of Modified Forms

• EMB1144 PTMs likely exist in substoichiometric amounts, making detection difficult against the background of unmodified protein.

• Solution: Use enrichment techniques such as phosphopeptide enrichment with titanium dioxide (TiO₂) or immunoprecipitation with PTM-specific antibodies before mass spectrometry analysis.

Challenge 2: Glycation Detection

• Advanced glycation end products (AGEs) can form on EMB1144 during stress conditions similar to other plant proteins .

• Solution: Use specific anti-CML (carboxymethyllysine) or anti-OGlcNAc antibodies (1:1000 and 1:5000 dilutions respectively) for Western blotting detection of glycated forms .

• Approach: Apply methods from glycation studies where samples are first separated on SDS-PAGE, followed by immunoblotting with anti-glycation antibodies.

Challenge 3: Mass Spectrometry Limitations

• PTMs can alter peptide behavior during ionization and fragmentation in MS/MS analysis.

• Solution: Use hybrid high-resolution LTQ/Orbitrap Velos instruments interfaced to nanoscale HPLC systems for improved detection sensitivity .

• Data Analysis: Apply specialized software like MaxQuant to analyze MS/MS data for PTMs, with appropriate statistical analysis using packages like DEP 1.1.4 to determine differential enrichment between treatments .

Challenge 4: Dynamic Nature of PTMs

• Redox-based modifications (e.g., disulfide bonds) can be reversible and change during sample preparation.

• Solution: Use rapid protein extraction methods with alkylating agents to preserve in vivo redox states, similar to approaches used in studying NPR1 oligomerization through intermolecular disulfide bonds .

Challenge 5: Validation of PTM Sites

• Confirming the exact residues modified requires site-directed mutagenesis.

• Solution: Generate point mutations at predicted modification sites and compare antibody reactivity between wild-type and mutant proteins.

Experimental Workflow for PTM Detection:

• Isolate EMB1144 using immunoprecipitation with specific antibodies

• Perform on-bead digestion with trypsin

• Analyze resulting peptides using high-resolution MS/MS

• Use specific enrichment techniques for targeted PTM detection

• Validate findings using immunoblotting with modification-specific antibodies

• Confirm biological relevance through mutagenesis of modification sites

This multifaceted approach is essential for comprehensive characterization of EMB1144 post-translational modifications under various developmental stages and stress conditions.

How can EMB1144 antibodies contribute to understanding biotin biosynthesis pathways?

EMB1144 antibodies can provide critical insights into the relationship between chorismate synthase function and biotin biosynthesis pathways in plants through several advanced research approaches:

Metabolic Crosstalk Investigation:

• While EMB1144 (chorismate synthase) and biotin biosynthesis operate in distinct pathways, their metabolic interactions can be studied using EMB1144 antibodies to examine how disruptions in aromatic amino acid synthesis affect biotin production.

• Research shows that embryo-defective mutants like bio1-1 and bio2-1 involved in biotin biosynthesis show similar phenotypes to emb mutants affecting chorismate synthase, suggesting potential metabolic interdependence .

Co-localization with Biotin Synthesis Enzymes:

• Use double immunolabeling with EMB1144 antibodies and antibodies against key biotin biosynthesis enzymes (BIO1, BIO2, BIO3-BIO1) to determine if these proteins co-localize in specific subcellular compartments or tissues.

• The bifunctional locus BIO3-BIO1 is particularly interesting as it represents a fusion of two biotin synthesis enzymes in Arabidopsis that could potentially interact with chorismate synthase networks .

Protein Complex Analysis:

• Employ co-immunoprecipitation with EMB1144 antibodies followed by mass spectrometry to identify potential interactions with biotin pathway components.

• Use reciprocal immunoprecipitation approaches to validate any identified interactions .

Developmental Regulation Studies:

• Compare the expression and localization patterns of EMB1144 and biotin synthesis enzymes during embryo development, particularly in relation to embryo-lethal phenotypes.

• Both pathways are essential for embryo development, with mutations leading to embryo arrest that can sometimes be rescued on enriched media .

Experimental Framework:

• Comparative Immunolocalization: Perform tissue and cellular localization studies of EMB1144 alongside biotin synthesis enzymes across developmental stages.

• Protein-Protein Interaction Analysis: Use techniques like proximity ligation assay (PLA) with EMB1144 antibodies and antibodies against biotin pathway components to detect potential proximity/interactions in situ.

• Metabolic Impact Assessment: Analyze how altered EMB1144 levels (in conditional mutants) affect biotin pathway enzyme levels and activities using immunoblotting and enzyme assays.

• Stress Response Correlation: Examine how abiotic stresses affect both pathways simultaneously by monitoring protein levels and modifications with specific antibodies.

These approaches can reveal previously unrecognized connections between aromatic amino acid biosynthesis via chorismate synthase and the biotin production pathway in plants, potentially uncovering new regulatory mechanisms important for embryo development.

What methodological approaches can resolve contradictory data regarding EMB1144 localization?

Resolving contradictory data regarding EMB1144 subcellular and tissue localization requires a systematic multi-method approach that addresses various technical limitations:

Fixation and Preparation Method Comparison

Different fixation protocols can significantly affect epitope accessibility and protein localization patterns. To resolve discrepancies:

• Parallel Processing Study: Compare chemical fixation (paraformaldehyde, glutaraldehyde) with cryofixation methods side-by-side.

• Antigen Retrieval Optimization: Test multiple antigen retrieval methods (heat-induced, enzymatic, pH-based) to determine optimal conditions for EMB1144 detection.

• Live Cell Imaging: When possible, combine antibody-based detection with live-cell approaches using fluorescently tagged EMB1144 to compare localizations.

Antibody Validation and Controls

• Multiple Antibody Approach: Use antibodies raised against different epitopes of EMB1144 to confirm localization patterns.

• Genetic Controls: Include EMB1144 knockout/knockdown tissues as negative controls and overexpression lines as positive controls.

• Peptide Competition Assays: Pre-incubate antibodies with immunizing peptides to confirm signal specificity.

• Western Blot Fractionation: Perform subcellular fractionation followed by Western blotting to biochemically verify localization patterns observed in microscopy.

Advanced Imaging Techniques

• Super-Resolution Microscopy: Apply techniques like structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) to resolve fine-scale localization beyond the diffraction limit.

• Electron Microscopy Immunogold Labeling: Use gold-conjugated secondary antibodies for ultrastructural localization studies with transmission electron microscopy.

• Correlative Light and Electron Microscopy (CLEM): Combine fluorescence microscopy with electron microscopy on the same sample to bridge resolution gaps.

Tissue-Specific and Developmental Analyses

• Developmental Time Course: Examine EMB1144 localization across multiple developmental stages, as localization may change dynamically.

• Cell Type-Specific Analysis: Compare localization patterns between different cell types (e.g., cortical vs. stelar tissues) to identify tissue-specific differences .

• Stress-Induced Relocalization: Examine whether abiotic stresses (e.g., salt stress) cause protein relocalization that might explain conflicting reports .

Computational Integration and Modeling

• Quantitative Colocalization Analysis: Use specialized software for statistical analysis of colocalization with established subcellular markers.

• Machine Learning Approaches: Apply advanced image analysis algorithms to detect subtle localization patterns across large datasets.

• Meta-analysis: Systematically compare methodologies across studies reporting conflicting localizations to identify procedural variables affecting outcomes.

By systematically applying these approaches and clearly documenting methodological details, researchers can resolve contradictory data regarding EMB1144 localization and establish a consensus view of its distribution in plant cells and tissues.

How does EMB1144 function relate to cell wall composition during plant development?

The relationship between EMB1144 function and cell wall composition represents a complex intersection of metabolic pathways with significant developmental implications:

Metabolic Connections:
EMB1144 (chorismate synthase) catalyzes a key step in the shikimate pathway, which produces precursors for:

• Aromatic amino acids (phenylalanine, tyrosine, tryptophan)

• Phenylpropanoid compounds essential for lignin biosynthesis

• Various secondary metabolites that contribute to cell wall structure and function

Transcriptomic studies reveal that genes involved in cell wall biosynthesis show coordinated expression patterns with EMB1144 during development and in response to environmental stresses .

Developmental Impact on Cell Wall Structure:

Tissue-Specific Relationships:

• In cortical tissues, EMB1144 expression correlates with distinctive cell wall composition, particularly in relation to pectin structure and water-soluble components .

• In stelar tissues, EMB1144 function appears more closely linked to secondary cell wall formation and lignification processes .

• Transcriptomic data shows that 17 water channel genes are down-regulated in cortical cells under salt stress, potentially affecting cell wall hydration and plasticity .

Stress Response Connections:

• Salt stress leads to significant down-regulation of multiple cell wall biosynthesis genes in cortical cells, correlating with altered EMB1144 expression .

• Specifically, salt stress affects the phenylpropanoid biosynthesis pathway in cortical cells, with multiple transcripts being down-regulated .

• The presence of distinct gelatinous layers with lamellar structure in bast fibers suggests a complex relationship between EMB1144 activity and specialized cell wall structures under stress conditions .

Research Methodologies for Investigation:

• Use EMB1144 antibodies to examine protein localization in relation to cell wall deposition patterns during development.

• Combine immunodetection with ultrastructural analyses to correlate EMB1144 presence with specific cell wall layers and structures.

• Employ conditional EMB1144 mutants to observe changes in cell wall composition through developmental stages.

• Analyze cell type-specific transcriptomes to identify coordinated expression between EMB1144 and cell wall biosynthesis genes.

This multifaceted relationship between EMB1144 function and cell wall composition underscores the importance of chorismate-derived metabolites in plant development and stress adaptation, revealing potential targets for enhancing crop resilience through cell wall modification.

What emerging technologies can enhance EMB1144 antibody-based research?

Emerging technologies are revolutionizing antibody-based research, offering new opportunities for studying EMB1144 with unprecedented precision and contextual information:

Advanced Spatial Transcriptomics/Proteomics Integration

Combining EMB1144 antibody detection with spatial transcriptomics enables simultaneous visualization of protein localization and associated gene expression patterns:

• Spatial Proteogenomics: Correlate EMB1144 protein distribution with transcript localization using techniques like Slide-seq or Visium spatial platforms.

• Single-Cell Antibody-Based Proteomics: Apply methods like CITE-seq (adapted for plant systems) to simultaneously detect EMB1144 protein and transcriptome in individual cells.

• Application: This integration would reveal how transcriptional regulation of EMB1144 and related genes translates to protein distribution across different cell types in plant tissues, building on previous cell type-specific transcriptional studies .

Proximity Labeling Technologies

These methods enable mapping of protein neighborhoods and transient interactions:

• TurboID and miniTurbo: These engineered biotin ligases fused to EMB1144 can biotinylate proximal proteins when expressed in plants, allowing subsequent purification and identification of the EMB1144 interactome.

• APEX2 Proximity Labeling: This peroxidase-based approach offers spatial and temporal control for mapping EMB1144 protein interactions under specific conditions.

• Application: These methods would expand on current knowledge of protein-protein interactions , revealing EMB1144's dynamic interaction partners during development and stress responses.

Cryo-Electron Tomography with Immunogold Labeling

This technology enables visualization of EMB1144 in its native cellular context:

• In situ Structural Analysis: Visualize EMB1144 within its cellular environment at near-atomic resolution.

• 3D Protein Complex Reconstruction: Determine the structure of EMB1144-containing complexes in their native state.

• Application: This approach would provide insights into how EMB1144 associates with cellular components, including the relationship between its localization and cell wall structures .

Nanobody and Single-Domain Antibody Technologies

These smaller antibody formats offer advantages for certain applications:

• Improved Penetration: Smaller size allows better tissue penetration and access to crowded cellular environments.

• Live-Cell Imaging: When fused to fluorescent proteins, anti-EMB1144 nanobodies enable real-time protein tracking in living plant cells.

• Application: These tools would facilitate studying the dynamics of EMB1144 localization during stress responses, such as salt stress adaptation .

Mass Spectrometry Imaging (MSI)

This technique allows spatial mapping of proteins and modifications:

• MALDI-Imaging MS: Maps EMB1144 distribution directly on tissue sections while preserving spatial information.

• PTM Mapping: Enables visualization of EMB1144 post-translational modifications across tissues.

• Application: MSI would complement antibody-based detection by providing label-free verification of EMB1144 distribution and modification state, particularly useful when studying glycation under stress conditions .

Microfluidic Antibody-Based Assays

These platforms enable high-throughput analysis with minimal sample requirements:

• Single-Cell Western Blotting: Analyze EMB1144 levels in individual cells isolated from different tissues.

• Microfluidic Immunoprecipitation: Perform IP-MS with microliter volumes of plant extracts.

• Application: These approaches would be particularly valuable for analyzing scarce samples, such as specific cell types isolated from plant tissues using techniques like FACS .

Implementation of these emerging technologies will significantly advance our understanding of EMB1144's role in plant development, stress responses, and metabolic pathways by providing more detailed, context-rich information about its expression, localization, interactions, and modifications.