At1g02190 Antibody

What is AT1G02190 and why would researchers develop antibodies against it?

AT1G02190 encodes a fatty acid hydroxylase superfamily protein in Arabidopsis thaliana that functions in oxidoreductase activity, iron ion binding, and catalytic activity. The protein is involved in oxidation reduction and fatty acid biosynthetic processes and is primarily localized to the endoplasmic reticulum. Its expression has been confirmed in twelve different plant structures across six growth stages, making it a significant component of plant development and metabolism . Researchers develop antibodies against AT1G02190 to study its expression patterns, subcellular localization, protein-protein interactions, and functional roles in fatty acid metabolism pathways. These antibodies serve as essential tools for understanding how this protein contributes to plant development, stress responses, and metabolic regulation. The development of highly specific antibodies enables researchers to distinguish AT1G02190 from closely related proteins, particularly important given that this protein belongs to a family with 2,538 blast hits across 532 species .

What types of antibodies can be generated against plant proteins like AT1G02190?

Researchers typically develop either polyclonal or monoclonal antibodies against plant proteins like AT1G02190, each with distinct advantages for different experimental applications. Polyclonal antibodies, similar to those developed against actin described in search result , are produced by immunizing rabbits or other host animals with recombinant proteins or synthetic peptides derived from AT1G02190. These antibodies recognize multiple epitopes on the target protein, potentially providing stronger signals in applications like Western blotting but with greater potential for cross-reactivity . Monoclonal antibodies, in contrast, are derived from a single B-cell clone and recognize a single epitope, offering higher specificity but potentially lower sensitivity. For membrane-bound proteins like AT1G02190, which is localized to the endoplasmic reticulum according to SUBAcon prediction with a confidence score of 1.000, researchers must carefully select immunogenic regions that are accessible while avoiding transmembrane domains . The choice between polyclonal and monoclonal approaches should be guided by the specific research objectives, with considerations for specificity, sensitivity, and the particular applications planned.

How can the specificity of antibodies against AT1G02190 be verified?

Verifying the specificity of antibodies against AT1G02190 requires a multi-faceted approach that combines molecular and biochemical techniques. The primary verification method involves Western blot analysis comparing wild-type Arabidopsis thaliana tissue with at1g02190 knockout or knockdown lines, expecting significantly reduced or absent signal in the mutant lines. Given that AT1G02190 has a calculated molecular weight of 72,096.50 Da , researchers should observe a band at approximately this size, with potential variations due to post-translational modifications. Competitive inhibition assays provide another verification approach, where pre-incubation of the antibody with purified recombinant AT1G02190 protein should abolish or significantly reduce the signal in subsequent immunoassays. Researchers should also test the antibody against related proteins, particularly AT2G37700.1, which TAIR10 identifies as the best Arabidopsis thaliana protein match to AT1G02190 . Immunoprecipitation followed by mass spectrometry can further confirm that the antibody specifically pulls down AT1G02190 rather than related proteins. Finally, immunolocalization studies should show endoplasmic reticulum localization, consistent with the SUBAcon prediction for this protein .

What are optimal strategies for designing immunogens for AT1G02190 antibody production?

Designing effective immunogens for AT1G02190 antibody production requires careful consideration of protein structure, accessibility, and uniqueness of epitopes. Based on the protein sequence data from TAIR10 , AT1G02190 has 627 amino acids with several domains including a fatty acid hydroxylase domain (InterPro:IPR006694) and a Wax2 C-terminal domain (InterPro:IPR021940). Researchers should avoid selecting immunogenic regions from highly conserved domains to minimize cross-reactivity with other fatty acid hydroxylase family members, particularly AT2G37700.1, which is the closest match in Arabidopsis . Instead, focus on unique regions with high antigenicity scores, preferably from hydrophilic portions of the protein that are likely to be exposed. For AT1G02190, the N-terminal region (amino acids 1-50) and C-terminal region (amino acids 577-627) often provide good candidates for peptide immunogens because they typically exhibit greater sequence diversity. The approach used for actin antibody production, where approximately 100 amino acids of recombinant protein conserved more than 80% across multiple forms were used as an immunogen , could be adapted by selecting regions of AT1G02190 that are less than 80% conserved among related proteins. For recombinant protein immunogens, expressing portions of AT1G02190 lacking transmembrane regions in E. coli or another expression system allows for production of soluble proteins that maintain native epitopes.

What expression systems are most effective for producing recombinant AT1G02190 for antibody generation?

The selection of an appropriate expression system for recombinant AT1G02190 production must balance protein yield, solubility, and preservation of native conformational epitopes. For plant membrane proteins like AT1G02190, which has membrane-associated domains and is localized to the endoplasmic reticulum , bacterial expression systems often result in inclusion bodies requiring solubilization and refolding. E. coli expression with fusion tags such as MBP (maltose-binding protein) or SUMO can improve solubility of selective domains, particularly the hydrophilic portions of the protein. For full-length expression, eukaryotic systems such as insect cells (Sf9 or High Five) using baculovirus vectors provide better membrane protein folding and post-translational modifications. Yeast expression systems like Pichia pastoris offer another alternative, combining relatively high yields with eukaryotic protein processing capabilities. Plant-based expression systems, including Nicotiana benthamiana transient expression or transgenic Arabidopsis, provide the most native environment for AT1G02190 expression, potentially preserving critical conformational epitopes. When working with AT1G02190, which has a calculated isoelectric point (pI) of 7.73 , buffer optimization during purification is essential to maintain protein stability. For antibody production purposes, expressing just the hydrophilic domains rather than the full-length protein often results in better immunogen quality while still generating antibodies capable of recognizing the native protein.

What purification techniques yield the highest quality AT1G02190 protein for immunization?

Purifying AT1G02190 for immunization requires approaches tailored to its biochemical properties, including its membrane association and calculated GRAVY score of -0.05, indicating moderate hydrophobicity . For recombinant protein fragments expressed with affinity tags, immobilized metal affinity chromatography (IMAC) using Ni-NTA or cobalt resins provides an effective initial purification step. Given AT1G02190's endoplasmic reticulum localization , purification protocols should incorporate detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin to solubilize membrane-associated regions while preserving protein structure. Size exclusion chromatography serves as a critical secondary purification step to separate monomeric protein from aggregates, which is particularly important for generating antibodies that recognize native conformations. For AT1G02190 with its calculated molecular weight of 72,096.50 Da , Superdex 200 columns typically provide optimal resolution. Ion exchange chromatography can further improve purity, with selection of cation or anion exchange depending on the expressed protein fragment's isoelectric point relative to the working pH. For immunization purposes, protein purity should exceed 90% as assessed by SDS-PAGE with Coomassie staining. Western blotting using anti-tag antibodies during purification development can confirm the identity of the purified protein, while mass spectrometry analysis provides ultimate verification of protein identity and purity before proceeding to immunization.

What immunization protocols maximize antibody titers against plant proteins like AT1G02190?

Developing high-titer antibodies against plant proteins such as AT1G02190 requires strategic immunization protocols that account for potential challenges in immunogenicity. Beginning with rabbits as host animals provides advantages for plant protein immunization due to their phylogenetic distance from plants, reducing the likelihood of tolerance to conserved epitopes. The initial immunization should include 250-500 μg of purified AT1G02190 protein or peptide conjugated to a carrier protein such as KLH (keyhole limpet hemocyanin) emulsified in complete Freund's adjuvant to stimulate a robust primary immune response. Subsequent booster immunizations at 21-day intervals should use incomplete Freund's adjuvant with 150-250 μg antigen, with a minimum of three boosters before the first test bleed. For challenging antigens like membrane proteins, alternative adjuvants such as TiterMax or Ribi can sometimes elicit stronger responses with less inflammation at immunization sites. Monitoring antibody titers via ELISA after each boost allows for optimization of the immunization schedule, continuing boosters until titers plateau. The approach used for actin antibodies, which resulted in highly specific polyclonal antibodies applicable across multiple plant species , could serve as a model for AT1G02190 antibody production. When targeting specific domains of AT1G02190, such as its fatty acid hydroxylase domain (InterPro:IPR006694) , researchers should consider immunizing multiple animals with different protein fragments to maximize the chances of obtaining high-quality antibodies against distinct epitopes.

How can AT1G02190 antibodies be used to study protein localization and trafficking in plant cells?

AT1G02190 antibodies enable detailed investigation of this protein's localization and trafficking pathways within plant cells through multiple complementary approaches. Immunofluorescence microscopy using fixed Arabidopsis tissues allows visualization of AT1G02190's native distribution, which should primarily show endoplasmic reticulum localization based on SUBAcon predictions with a confidence score of 1.000 . This technique requires careful optimization of fixation conditions and antibody dilutions, with recommendations starting at 1:100-1:250 dilution ratios similar to those used for actin antibodies in immunofluorescence applications . For higher-resolution analysis, expansion microscopy (ExM) significantly improves visualization of subcellular structures, with AT1G02190 antibodies used at approximately 1:250 dilution as suggested for similar plant protein studies . Immunogold electron microscopy provides nanometer-resolution localization, critical for distinguishing between closely positioned organelles like the endoplasmic reticulum and Golgi apparatus. For studying dynamic trafficking, researchers can combine AT1G02190 antibodies with live-cell imaging of fluorescently tagged markers for different endomembrane compartments. Co-immunoprecipitation experiments using AT1G02190 antibodies followed by mass spectrometry can identify interacting proteins involved in its trafficking, potentially revealing regulatory mechanisms controlling its movement between compartments. Researchers should validate antibody specificity in each application through appropriate controls, including comparison with AT1G02190-fluorescent protein fusions and testing in knockout/knockdown lines.

What approaches can integrate AT1G02190 antibodies with proteomics for studying protein interactions?

Integrating AT1G02190 antibodies with proteomics technologies enables comprehensive mapping of protein interaction networks and post-translational modifications. Co-immunoprecipitation (Co-IP) using AT1G02190 antibodies conjugated to protein A/G beads or directly to agarose/magnetic beads serves as the foundation for interaction studies, capturing both direct binding partners and components of larger complexes. Following Co-IP, mass spectrometry analysis can identify the full complement of interacting proteins, with experimental designs including SILAC (Stable Isotope Labeling with Amino acids in Cell culture) or TMT (Tandem Mass Tag) labeling to provide quantitative comparison across different conditions. Proximity-dependent biotin identification (BioID) coupled with AT1G02190 antibodies offers another approach, where proteins spatially close to AT1G02190 become biotinylated and can be purified using streptavidin before mass spectrometry analysis. For studying dynamic changes in AT1G02190 interaction networks during plant development or stress responses, time-course experiments sampling multiple stages and conditions should be conducted. Cross-linking mass spectrometry (XL-MS) provides more detailed structural information by capturing transient interactions through chemical cross-linkers before immunoprecipitation with AT1G02190 antibodies. For confirming high-confidence interactions identified through these proteomics approaches, reciprocal Co-IP experiments and yeast two-hybrid assays provide orthogonal validation, strengthening confidence in the biological relevance of the interactions.

What experimental designs best utilize AT1G02190 antibodies to investigate its role in fatty acid biosynthesis?

Investigating AT1G02190's role in fatty acid biosynthesis requires strategic experimental designs that leverage antibodies to connect molecular mechanisms with physiological outcomes. Immunoprecipitation followed by activity assays represents a powerful approach, where AT1G02190 is captured from plant extracts using specific antibodies and then assessed for fatty acid hydroxylase activity using appropriate substrates and analytical methods like gas chromatography-mass spectrometry (GC-MS). To establish causality between AT1G02190 expression levels and fatty acid profiles, researchers should design experiments comparing wild-type plants with AT1G02190 knockout/knockdown lines and overexpression lines, using the antibodies to confirm protein expression levels via Western blotting at 1:3000-1:5000 dilution ratios similar to those used for other plant proteins . Chromatin immunoprecipitation (ChIP) experiments using antibodies against transcription factors potentially regulating AT1G02190 can connect upstream regulatory networks to fatty acid metabolism. For dissecting AT1G02190's interactions with other components of the fatty acid biosynthetic pathway, co-immunoprecipitation with AT1G02190 antibodies followed by targeted Western blotting or mass spectrometry can identify stable protein complexes. Time-course experiments examining AT1G02190 expression, localization, and interaction partners during developmental stages with active fatty acid synthesis provide crucial temporal context. Metabolic labeling studies using radioactive or stable isotope-labeled fatty acid precursors, combined with immunoprecipitation of AT1G02190, can directly connect the protein to specific steps in the biosynthetic pathway through the analysis of labeled intermediates associated with the immunoprecipitated protein.

What are common sources of non-specific binding when using AT1G02190 antibodies in immunoassays?

Non-specific binding in AT1G02190 antibody applications can arise from multiple sources that researchers must systematically address. Cross-reactivity with closely related proteins represents a primary concern, particularly with AT2G37700.1, which TAIR10 identifies as the best Arabidopsis thaliana protein match to AT1G02190 . This issue becomes particularly significant in polyclonal antibody preparations, which recognize multiple epitopes and may bind conserved regions shared across the fatty acid hydroxylase superfamily. Researchers should pre-clear antibody preparations by adsorption against recombinant related proteins or tissue extracts from AT1G02190 knockout plants. Protein A/G purification of IgG fractions from serum can significantly reduce background by eliminating non-specific serum proteins, similar to purification approaches used for other plant protein antibodies . For Western blotting applications, increasing blocking agent concentration (5% non-fat dry milk or BSA) and adding 0.1-0.3% Tween-20 to wash buffers helps reduce hydrophobic interactions. In immunohistochemistry and immunofluorescence, autofluorescence from phenolic compounds, chlorophyll, and cell wall components often creates false positives; this can be mitigated through appropriate quenching steps and careful selection of fluorophores with emission spectra distinct from plant autofluorescence. The inclusion of additional blocking agents such as normal serum from the same species as the secondary antibody (5-10%) can prevent secondary antibody non-specific binding. Quantitative analysis of signal-to-noise ratios across different antibody dilutions and blocking conditions should guide optimization for each specific application.

What statistical approaches best validate immunoblot quantification for AT1G02190 expression studies?

Rigorous statistical validation of immunoblot quantification ensures reliable interpretation of AT1G02190 expression data across experimental conditions. Researchers should implement appropriate normalization strategies, using housekeeping proteins such as actin, detected with validated antibodies at optimized dilutions (1:3000-1:5000) , as loading controls. Given that AT1G02190 has a calculated molecular weight of 72,096.50 Da , researchers should carefully select normalization proteins that do not co-migrate on gels, avoiding potential signal overlap. For each experiment, technical replicates (minimum of three) and biological replicates (typically 3-5 independent samples) provide the foundation for statistical analysis. Densitometric analysis should utilize specialized software capable of background subtraction and accurate band integration, with linear regression analysis of serial dilutions confirming the quantitative range of detection for both AT1G02190 and reference proteins. Statistical analysis using ANOVA followed by appropriate post-hoc tests (Tukey's HSD for comparing multiple groups or Dunnett's test when comparing against a control) identifies significant differences between experimental conditions. For complex experimental designs examining AT1G02190 expression across multiple conditions and time points, mixed-effects models provide more appropriate statistical frameworks that account for both fixed and random effects. Power analysis before experimentation helps determine the minimum sample size needed to detect biologically meaningful changes in AT1G02190 expression with statistical confidence. Researchers should report both normalized band intensities and measurement uncertainty (standard deviation or standard error) in publications, along with specific p-values rather than arbitrary significance thresholds, providing transparent and reproducible quantification of AT1G02190 expression.

How do researchers address challenges in developing antibodies against highly conserved regions of AT1G02190?

Developing antibodies against highly conserved regions of AT1G02190 presents significant challenges that require specialized approaches to achieve specificity. The computational description from TAIR10 indicates that AT1G02190 contains InterPro domains such as Fatty acid hydroxylase (IPR006694) that may be conserved across multiple species . When targeting such conserved regions, researchers should first perform comprehensive sequence alignment analysis across related proteins in Arabidopsis and other species to identify subtle amino acid differences that can be exploited for specificity. Modification of conserved peptide immunogens through strategic substitution of non-essential amino acids with non-native residues can enhance immunogenicity while maintaining the ability to recognize the native protein. For recombinant protein immunogens, expressing chimeric proteins that combine conserved domains of AT1G02190 with unrelated carrier proteins often improves immune responses against the target epitopes. Post-immunization antibody purification techniques such as affinity chromatography using immobilized AT1G02190-specific peptides can enrich for antibodies recognizing the desired epitopes while removing those binding to highly conserved regions. Negative selection approaches, where antibody preparations are passed through columns containing related proteins to remove cross-reactive antibodies, further improve specificity. These strategies have been successfully applied to other plant proteins, as demonstrated by the development of actin antibodies that specifically recognize multiple actin isoforms while maintaining high specificity . For the most challenging conserved epitopes, developing monoclonal antibodies with extensive screening for clones recognizing specific variants of the conserved region offers another viable approach.

How can AT1G02190 antibodies be integrated with CRISPR/Cas9 gene editing for functional studies?

Integrating AT1G02190 antibodies with CRISPR/Cas9 gene editing technologies creates powerful experimental systems for dissecting protein function with unprecedented precision. CRISPR/Cas9-generated knockout lines provide essential negative controls for antibody validation, confirming specificity by demonstrating absence of signal in Western blots, immunoprecipitation, and immunofluorescence experiments. For more sophisticated functional studies, researchers can use CRISPR/Cas9 to introduce precise mutations in specific domains of AT1G02190, such as the fatty acid hydroxylase domain (InterPro:IPR006694) or the Wax2 C-terminal domain (InterPro:IPR021940) , then use antibodies to confirm expression of the mutant protein and analyze changes in localization, interaction partners, or post-translational modifications. Epitope tagging through CRISPR/Cas9-mediated homology-directed repair allows introduction of small epitope tags that don't disrupt protein function, enabling comparison between antibody detection of native AT1G02190 and tag-based detection systems. For studying essential genes where knockout is lethal, CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) systems modulate expression levels without complete ablation, with antibodies providing precise quantification of the resulting protein levels. Antibodies against AT1G02190 can also help evaluate potential off-target effects of CRISPR/Cas9 editing by confirming that expression levels of closely related proteins remain unchanged. Temporal control systems like chemically-inducible or optogenetic CRISPR/Cas9 variants allow precise timing of gene disruption, with antibodies tracking the subsequent decay of existing protein to establish the temporal window of depletion, critical for connecting phenotypic changes to protein absence rather than secondary effects.

What comparative analytical approaches reveal insights when studying AT1G02190 across different plant species?

Cross-species analysis of AT1G02190 using antibody-based approaches provides evolutionary and functional insights that cannot be obtained from single-species studies. AT1G02190 antibodies with demonstrated cross-reactivity, similar to actin antibodies that recognize conserved epitopes across multiple plant species , enable comparative studies of protein expression, localization, and function. Depending on epitope conservation, researchers may need to test AT1G02190 antibodies across phylogenetically diverse plant species including model systems like Arabidopsis thaliana, crop species in the Brassicaceae family, and more distant relatives. Western blot analysis comparing AT1G02190 homologs across species can reveal differences in expression levels, molecular weight (due to species-specific post-translational modifications), and tissue-specific expression patterns. Immunoprecipitation followed by mass spectrometry across multiple species identifies conserved and species-specific interaction partners, highlighting evolutionarily conserved functional complexes versus specialized adaptations. Quantitative proteomics approaches using isobaric tags for relative and absolute quantification (iTRAQ) or tandem mass tags (TMT) provide precise comparative expression data across species under identical experimental conditions. Immunohistochemistry and immunofluorescence studies across species reveal conservation or divergence in subcellular localization patterns, with particular attention to whether the endoplasmic reticulum localization predicted for Arabidopsis AT1G02190 is maintained across species. Collaborative research networks analyzing AT1G02190 across multiple species under standardized conditions using the same validated antibodies can generate comprehensive datasets revealing how this protein's function has evolved across plant lineages, connecting molecular differences to physiological adaptations in fatty acid metabolism.