At4g12490 Antibody

What is AT4G12490 and why is it significant in plant research?

AT4G12490 encodes a bifunctional inhibitor/lipid-transfer protein that plays important roles in plant development and stress responses. It has been identified as one of the differentially expressed genes in studies of Casparian strip formation, which is critical for controlling nutrient and water uptake in plant roots . Additionally, AT4G12490 appears as an up-regulated gene in studies involving BAK1 (BRI1-associated receptor kinase), a central regulator in multiple plant signaling pathways . Understanding this protein's function provides insights into fundamental plant biology mechanisms including barrier formation and defense responses. The protein's dual functionality as both an inhibitor and lipid-transfer molecule makes it particularly interesting for studying plant cell wall development and responses to environmental stresses.

What are the optimal sample preparation methods for AT4G12490 antibody experiments?

For optimal results with AT4G12490 antibodies, sample preparation should focus on preserving protein structure while minimizing contamination. Start with fresh plant tissue extraction using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail. For immunohistochemistry applications, fixation with 4% paraformaldehyde followed by embedding in paraffin or resin works effectively for localization studies. When working with root tissues where Casparian strip formation occurs, consider using longitudinal sections to visualize the endodermal layer properly. Protein extraction should be performed at 4°C to prevent degradation, and samples should be processed immediately or stored at -80°C for later analysis. For immunoprecipitation experiments, crosslinking with formaldehyde before extraction can help preserve protein-protein interactions that may be relevant to AT4G12490 function.

How can I validate the specificity of an AT4G12490 antibody?

Validating antibody specificity is crucial for reliable research outcomes. For AT4G12490 antibodies, implement multiple validation approaches. First, perform Western blot analysis with both wild-type plant extracts and AT4G12490 knockout mutants, expecting signal absence in the mutant. Second, conduct competitive blocking experiments using recombinant AT4G12490 protein to confirm binding specificity. Third, employ immunoprecipitation followed by mass spectrometry to verify the antibody captures the intended target. Western blot analysis should reveal a protein band at the expected molecular weight (approximately 10-15 kDa for most lipid transfer proteins). Additionally, sequence- and modification-specific antibodies can be developed following approaches similar to those used for BAK1 studies, where phosphorylation-specific antibodies were created to monitor specific modification sites .

What controls should be included in immunohistochemistry experiments with AT4G12490 antibodies?

When performing immunohistochemistry with AT4G12490 antibodies, several controls are essential for result validation. Include a negative control using pre-immune serum or isotype-matched control antibodies to assess background staining. Include tissue from AT4G12490 knockout or knockdown plants as a biological negative control. For positive controls, use tissues known to express high levels of AT4G12490, such as roots where Casparian strip formation occurs. If available, include a blocking peptide control where the primary antibody is pre-incubated with excess recombinant AT4G12490 protein, which should eliminate specific staining. Additionally, perform parallel staining with antibodies against known endodermal markers to confirm localization patterns in relation to Casparian strip formation. These comprehensive controls will help distinguish specific from non-specific binding and validate experimental findings.

How should I design experiments to study AT4G12490 expression changes during stress responses?

To effectively study AT4G12490 expression changes during stress responses, implement a time-course experimental design with multiple stress conditions. Begin with a baseline measurement in standard growth conditions, then expose plants to various stresses (drought, salt, pathogen exposure, or nutrient deficiency) and collect samples at regular intervals (0, 1, 3, 6, 12, 24, and 48 hours). For each timepoint, extract both RNA for transcript analysis (RT-qPCR) and protein for Western blot analysis using the AT4G12490 antibody. Include appropriate housekeeping genes and proteins as internal controls. Incorporate multiple biological and technical replicates to ensure statistical robustness. For more comprehensive insights, consider complementing antibody-based techniques with reporter gene constructs (e.g., AT4G12490 promoter:GUS) to visualize spatial expression patterns. Compare expression patterns with known stress-responsive genes to contextualize AT4G12490's role in stress response networks.

What are the recommended protocols for using AT4G12490 antibodies in immunoprecipitation experiments?

For effective immunoprecipitation of AT4G12490 and its interaction partners, begin with 2-3 g of fresh plant tissue extracted in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, phosphatase inhibitors, and protease inhibitors. Pre-clear the lysate by incubation with Protein A/G beads for 1 hour at 4°C. Incubate the pre-cleared lysate with 2-5 μg of AT4G12490 antibody overnight at 4°C with gentle rotation. Add fresh Protein A/G beads and incubate for an additional 3 hours. Wash the beads at least 4 times with extraction buffer containing reduced detergent concentration (0.1% Triton X-100). For co-immunoprecipitation studies, elute bound proteins with a low-pH glycine buffer or by boiling in SDS sample buffer. For chromatin immunoprecipitation (if applicable), include a crosslinking step with 1% formaldehyde before extraction. Always include appropriate controls, including IgG antibody control and input samples, to accurately assess enrichment.

How can I optimize Western blot conditions for AT4G12490 antibody detection?

Optimizing Western blot conditions for AT4G12490 antibody detection requires careful attention to several parameters. First, because AT4G12490 is a relatively small protein (approximately 10-15 kDa), use higher percentage (15-18%) SDS-PAGE gels for better resolution of lower molecular weight proteins. Transfer proteins to PVDF membrane (rather than nitrocellulose) using a wet transfer system at 30V overnight at 4°C for efficient transfer of small proteins. Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Optimize primary antibody concentration through titration (typically starting at 1:1000 and adjusting as needed) and incubate overnight at 4°C. Use a high-sensitivity detection system such as enhanced chemiluminescence or fluorescent secondary antibodies for visualization. If background is an issue, increase washing steps (5-6 washes with TBST for 10 minutes each) and consider using more stringent blocking agents or adding 0.05% SDS to the antibody dilution buffer to reduce non-specific binding.

What approaches can detect post-translational modifications of AT4G12490 protein?

Detecting post-translational modifications (PTMs) of AT4G12490 requires specialized approaches. First, develop or obtain modification-specific antibodies that recognize particular PTMs, similar to the phosphorylation-specific antibodies developed for BAK1 studies . For phosphorylation analysis, immunoprecipitate AT4G12490 using the general antibody, then probe with phospho-specific antibodies (anti-phosphoserine, anti-phosphothreonine, or anti-phosphotyrosine) on Western blots. Alternatively, perform phosphatase treatment on half of your protein sample and compare with untreated samples to identify mobility shifts. For comprehensive PTM mapping, use mass spectrometry approaches after immunoprecipitation with the AT4G12490 antibody. Enrichment strategies for specific modifications include titanium dioxide for phosphopeptides or lectin affinity chromatography for glycosylated forms. Consider using Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms based on mobility differences. For in vivo studies, combine these approaches with treatments that affect specific kinases or phosphatases to understand the regulation of AT4G12490 modifications.

How can AT4G12490 antibodies be used to study protein-protein interactions in Casparian strip formation?

AT4G12490 antibodies provide powerful tools for investigating protein-protein interactions in Casparian strip formation through multiple sophisticated approaches. Implement proximity-dependent labeling techniques like BioID or APEX2, where AT4G12490 is fused to a biotin ligase or peroxidase and expressed in plants, followed by streptavidin pulldown and identification of proximal proteins using mass spectrometry. This approach can be complemented with traditional co-immunoprecipitation using AT4G12490 antibodies followed by mass spectrometry to identify interaction partners. For visualization of interactions in situ, perform proximity ligation assays (PLA) using the AT4G12490 antibody paired with antibodies against suspected interaction partners, generating fluorescent signals when proteins are within 40 nm of each other. To validate direct interactions, use yeast two-hybrid or split-GFP assays with candidate partners identified through these initial screens. Focus on interactions with known Casparian strip proteins like ESB1 and CASP family proteins to place AT4G12490 within the functional network regulating barrier formation. These multi-faceted approaches will provide complementary evidence for the protein's role in Casparian strip development .

What are the challenges in developing highly specific antibodies against the AT4G12490 protein family?

Developing highly specific antibodies against AT4G12490 presents several significant challenges. First, the protein belongs to a family of lipid transfer proteins with high sequence similarity among members, making it difficult to find unique epitopes for antibody generation. Second, the protein's small size (10-15 kDa) limits the number of potential antigenic regions. Third, different post-translational modifications may affect antibody recognition, potentially leading to inconsistent results across different plant tissues or conditions. To overcome these challenges, implement a comprehensive epitope mapping strategy to identify unique regions of AT4G12490 not conserved in related proteins. Consider using recombinant protein expression systems that maintain proper folding and modifications as immunogens. Alternatively, use synthetic peptides from unique regions, coupled to carrier proteins for immunization. Employ advanced antibody engineering approaches like those described for designing antibodies with customized specificity profiles, which can discriminate between very similar epitopes . Extensive validation using knockout plants, competitive binding assays, and cross-reactivity tests against related proteins is essential to ensure specificity before use in research applications.

How can AT4G12490 antibodies help understand the differential regulation of gene expression in BAK1 signaling pathways?

AT4G12490 antibodies can provide critical insights into BAK1 signaling pathways by facilitating multi-layered analysis of protein expression changes. Since AT4G12490 is among the up-regulated genes in BAK1(Y610F)-Flag plants , changes in its protein levels may serve as a downstream readout of altered BAK1 signaling. Design comparative studies between wild-type, BAK1-Flag, and BAK1(Y610F)-Flag plants under various conditions, including pathogen-associated molecular pattern (PAMP) exposure or brassinosteroid treatments. Use AT4G12490 antibodies for Western blot analysis to quantify protein abundance changes in response to these treatments. Combine this with chromatin immunoprecipitation (ChIP) using antibodies against transcription factors that might regulate AT4G12490 expression, such as WRKY71, which appears in similar datasets . For comprehensive pathway analysis, perform immunoprecipitation of BAK1 followed by mass spectrometry to identify interaction partners that might link BAK1 signaling to AT4G12490 regulation. Create reporter constructs with the AT4G12490 promoter to visualize expression patterns in different BAK1 genetic backgrounds. This integrated approach will help decipher how AT4G12490 fits into the complex regulatory networks governed by BAK1 in plant immunity and development.

How might co-localization studies with AT4G12490 antibodies inform our understanding of subcellular protein trafficking?

Co-localization studies using AT4G12490 antibodies can reveal crucial information about protein trafficking pathways relevant to plant cell wall development and defense responses. Implement high-resolution confocal microscopy with dual immunofluorescence labeling using AT4G12490 antibodies alongside markers for different cellular compartments (endoplasmic reticulum, Golgi apparatus, plasma membrane, cell wall). For dynamic trafficking studies, combine this with treatments that disrupt specific trafficking pathways, such as Brefeldin A (inhibits ER-to-Golgi transport) or wortmannin (affects vacuolar targeting). Enhance spatial resolution with super-resolution microscopy techniques like structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy to precisely map AT4G12490 localization relative to cell wall reinforcement sites during Casparian strip formation. For temporal insights, perform time-course studies during pathogen challenge or abiotic stress exposure to track changes in AT4G12490 localization. Combine immunolocalization with transmission electron microscopy for ultrastructural localization at the cell wall-plasma membrane interface. These approaches will help determine whether AT4G12490 participates in secretory pathways for cell wall component deposition, potentially revealing new mechanisms in barrier formation and plant defense responses.

How should researchers interpret contradictory immunolocalization results with AT4G12490 antibodies?

When confronting contradictory immunolocalization results with AT4G12490 antibodies, adopt a systematic troubleshooting and validation approach. First, assess antibody specificity through comprehensive testing against wild-type and knockout plants using Western blots. Second, evaluate fixation and permeabilization methods, as these can dramatically affect epitope accessibility and result in false negative or altered localization patterns. Third, consider tissue-specific or developmental stage-specific expression differences that might explain variable results. Fourth, examine potential post-translational modifications or protein-protein interactions that might mask epitopes in specific cellular contexts. When analyzing contradictory results, perform side-by-side comparisons using multiple antibodies targeting different epitopes of AT4G12490. Supplement antibody-based approaches with fluorescent protein fusions to corroborate localization patterns. Quantify signal intensities across different experimental conditions to detect subtle differences in localization patterns. Finally, cross-reference results with publicly available transcriptomic and proteomic datasets to establish expected expression patterns. This multi-faceted approach will help distinguish technical artifacts from biologically meaningful variations in AT4G12490 localization.

What statistical approaches are recommended for quantifying AT4G12490 expression changes in immunoblot studies?

For rigorous quantification of AT4G12490 expression changes in immunoblot studies, implement comprehensive statistical approaches. Begin with densitometric analysis of band intensities using software such as ImageJ, normalizing AT4G12490 signals to appropriate loading controls (e.g., actin, tubulin, or total protein staining with Ponceau S). Perform at least three biological replicates and multiple technical replicates to ensure robust statistical analysis. Apply logarithmic transformation to densitometry data if it shows heteroscedasticity. For comparison between two conditions, use paired t-tests; for multiple conditions, use one-way ANOVA followed by appropriate post-hoc tests such as Tukey's HSD. When analyzing time-series data, consider repeated measures ANOVA or mixed-effects models. For studies comparing mutant lines with subtle differences (as seen in the BAK1/Y610F comparisons), perform power analysis to ensure sufficient sample size for detecting potentially small but meaningful differences. Report fold changes with confidence intervals rather than just p-values, and consider using non-parametric tests if data do not meet normality assumptions. This comprehensive statistical approach will increase the reliability and reproducibility of AT4G12490 expression studies.

How can researchers distinguish between specific signal and background when using AT4G12490 antibodies in plant tissues with high autofluorescence?

Distinguishing specific antibody signal from background in plant tissues requires specialized techniques to overcome the challenge of autofluorescence. First, implement spectral unmixing during confocal microscopy, where the distinct spectral signatures of autofluorescence and fluorophore-conjugated antibodies are computationally separated. Second, use fluorophores with emission spectra distinct from plant autofluorescence (far-red dyes like Alexa Fluor 647 or Cy5 are particularly effective). Third, employ autofluorescence quenching methods such as treating sections with Sudan Black B (0.1% in 70% ethanol) or sodium borohydride (0.1% in PBS) before antibody incubation. Fourth, include appropriate technical controls: (1) no-primary antibody control, (2) isotype control, and (3) competitive blocking with recombinant AT4G12490. Fifth, acquire images of untreated tissue to establish baseline autofluorescence patterns for comparison. For quantitative analysis, use signal-to-noise ratio measurements rather than absolute intensity values. When designing experiments, consider using transgenic plants expressing fluorescent protein-tagged AT4G12490 as alternative or complementary approaches to antibody-based detection, allowing for live-cell imaging without fixation-induced artifacts.

How should researchers interpret AT4G12490 expression data in the context of BAK1-mediated signaling pathways?

Interpreting AT4G12490 expression data in BAK1-mediated signaling pathways requires careful consideration of experimental context and complementary datasets. Based on available data, AT4G12490 appears up-regulated in BAK1(Y610F)-Flag plants with a fold change of 0.43 compared to BAK1-Flag plants . This suggests potential involvement in BAK1-mediated pathways, which include both immune and developmental responses. When analyzing such data, first verify expression changes using multiple techniques (RT-qPCR, Western blot, reporter gene assays) to ensure consistency. Consider that low abundance transcripts may show poor correlation between microarray and qPCR results, as noted in the BAK1 study . Place AT4G12490 expression changes in broader context by examining co-regulated genes and performing pathway enrichment analysis. Since BAK1 participates in both brassinosteroid signaling and pathogen-associated molecular pattern (PAMP)-triggered immunity, determine which pathway predominantly affects AT4G12490 through treatments with brassinosteroids or PAMPs like flg22. For definitive pathway assignment, examine AT4G12490 expression in additional mutant backgrounds affecting specific branches of BAK1 signaling. This integrated approach will help position AT4G12490 correctly within the complex signaling networks regulated by BAK1.

How can AT4G12490 antibodies be used in high-throughput screening of plant stress responses?

AT4G12490 antibodies can be adapted for high-throughput screening of plant stress responses through several innovative approaches. Develop antibody-based ELISA assays in 96-well format for rapid quantification of AT4G12490 protein levels across multiple samples and conditions. This system can screen hundreds of Arabidopsis accessions or mutant lines for differential AT4G12490 expression in response to standardized stress treatments. Alternatively, create protein microarrays where plant extracts from different treatments are spotted onto slides and probed with AT4G12490 antibodies, allowing for parallel analysis of thousands of samples. For even higher throughput, implement automated immunohistochemistry platforms using tissue microarrays containing multiple plant samples. Combine these approaches with image-based phenotyping platforms to correlate AT4G12490 expression with morphological stress responses. To increase information yield, develop multiplexed assays using differently labeled antibodies against AT4G12490 and other stress-responsive proteins. These high-throughput approaches can be particularly valuable for screening chemical libraries for compounds that modulate AT4G12490 expression, potentially identifying new tools for agricultural applications in stress resistance.

What are the potential applications of AT4G12490 antibodies in studying plant-pathogen interactions?

AT4G12490 antibodies offer versatile tools for investigating plant-pathogen interactions across multiple experimental systems. Since AT4G12490 appears related to plant defense responses (based on its differential regulation in BAK1 signaling contexts ), antibodies can track its expression dynamics during pathogen infection time courses. Implement immunohistochemistry to visualize AT4G12490 localization at infection sites, particularly at cell wall reinforcement areas where pathogen penetration is resisted. Use immunoprecipitation followed by mass spectrometry to identify pathogen-induced changes in AT4G12490 interaction partners or post-translational modifications that might regulate its activity during defense responses. For functional studies, combine antibody-based protein depletion approaches (through intrabodies or immunoprecipitation) with pathogen challenge assays to determine AT4G12490's necessity in resistance. Given that several genes down-regulated in BAK1(Y610F)-Flag plants are involved in defense responses , investigate whether AT4G12490 functions antagonistically to these defense genes or in parallel pathways. Develop assays to determine if AT4G12490's potential lipid-transfer activity is modulated during pathogen attack, possibly affecting the composition of barrier lipids at infection sites.

How might custom-designed antibodies against specific AT4G12490 epitopes advance our understanding of protein function?

Developing custom-designed antibodies against specific AT4G12490 epitopes can significantly advance functional studies through targeted approaches. By applying inference and design strategies similar to those described for antibody specificity in search result , researchers can create antibodies that specifically recognize distinct functional domains of AT4G12490. For instance, generate domain-specific antibodies targeting the lipid-binding pocket versus the inhibitory domain to differentiate these functions in various contexts. Design modification-specific antibodies that recognize post-translationally modified forms (phosphorylated, glycosylated, etc.) to study regulation mechanisms. Create conformation-specific antibodies that distinguish between active and inactive protein states, enabling visualization of activation patterns during stress responses or development. These specialized antibodies can reveal when and where specific protein domains are accessible or modified, providing insights into regulatory mechanisms. Additionally, develop antibodies that can distinguish between AT4G12490 and closely related family members, allowing for precise study of this specific protein despite sequence similarities with other lipid transfer proteins. This approach would benefit from computational modeling to identify unique epitopes and predict antibody-antigen interactions before experimental validation.

What novel approaches could integrate AT4G12490 antibody-based detection with other -omics technologies?

Integrating AT4G12490 antibody-based detection with cutting-edge -omics technologies can provide unprecedented insights into protein function within broader biological networks. Combine immunoprecipitation using AT4G12490 antibodies with RNA-seq (RIP-seq) to identify RNAs that might associate with this protein, potentially revealing unexpected roles in RNA metabolism or transport. Integrate antibody-based spatial proteomics approaches, such as proximity labeling followed by mass spectrometry, with transcriptomic data to correlate spatial protein interactions with gene expression patterns. Implement single-cell approaches by adapting AT4G12490 antibodies for mass cytometry (CyTOF) to quantify protein levels alongside dozens of other markers at single-cell resolution in plant tissues. For integration with metabolomics, perform immunoprecipitation of AT4G12490 followed by analysis of co-purified lipids or other small molecules to directly connect the protein with its potential lipid transport substrates. Develop computational frameworks that integrate antibody-based protein quantification data with transcriptome, metabolome, and phenome datasets to build comprehensive models of AT4G12490 function in plant development and stress responses. These multi-omics approaches will position AT4G12490 within its broader functional context and potentially reveal unexpected roles beyond current annotations.