At3g15700 Antibody

What is the At3g15700 protein and why is it significant in plant research?

The At3g15700 protein is a putative disease resistance protein found in Arabidopsis thaliana (mouse-ear cress), a widely used model organism in plant biology. It consists of 375 amino acids with a molecular mass of approximately 42.352 kDa . The protein's significance lies in its potential role in plant immunity and disease resistance mechanisms, making it an important target for researchers studying plant-pathogen interactions and immune response pathways . Understanding At3g15700 contributes to our broader knowledge of how plants defend themselves against pathogens, which has implications for agricultural applications and crop protection strategies. Similar to other disease resistance proteins in Arabidopsis, At3g15700 likely plays a role in the plant's innate immune system, potentially recognizing pathogen-derived molecules and triggering downstream defense responses.

What are the key specifications of commercially available At3g15700 antibodies?

Commercial At3g15700 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana At3g15700 protein . These antibodies are available in liquid form with storage buffers containing preservatives like 0.03% Proclin 300 and constituents such as 50% glycerol and 0.01M PBS at pH 7.4 . They are purified using antigen affinity purification methods to ensure specificity and quality . The antibodies are designed for research applications including ELISA and Western blotting for identifying the target antigen . It's important to note that these antibodies are specifically designated "For Research Use Only" and not intended for diagnostic or therapeutic procedures . The lead time for made-to-order At3g15700 antibodies is typically 14-16 weeks, which researchers should factor into their experimental timelines .

What storage and handling protocols are recommended for maintaining At3g15700 antibody activity?

To maintain optimal At3g15700 antibody activity, proper storage and handling protocols are essential. Upon receipt, store the antibody at -20°C or -80°C to preserve its structural integrity and binding capacity . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of antibody function . When working with the antibody, aliquot it into smaller volumes before freezing to minimize freeze-thaw cycles. For short-term use, the antibody can be stored at 4°C for up to one week, but long-term storage requires freezing temperatures. The antibody is typically provided in a storage buffer containing 50% glycerol and 0.03% Proclin 300 preservative in PBS (pH 7.4), which helps maintain stability . When diluting the antibody for experimental procedures, use fresh buffer systems and maintain sterile conditions to prevent microbial contamination. Additionally, avoid exposing the antibody to strong light or extreme pH conditions, as these factors can compromise its activity.

How can At3g15700 antibody specificity be validated in experimental systems?

Validating the specificity of At3g15700 antibody is crucial for ensuring reliable experimental results. Multiple approaches should be implemented for comprehensive validation. First, perform Western blot analysis using both wild-type Arabidopsis thaliana lysates and At3g15700 knockout/knockdown lines to confirm the absence or reduction of signal in the latter. Second, conduct pre-absorption tests by incubating the antibody with excess purified recombinant At3g15700 protein before immunostaining or Western blotting to demonstrate signal reduction. Third, verify target molecular weight (approximately 42.352 kDa) in immunoblots . Fourth, compare results with alternative antibodies targeting different epitopes of the same protein when available. For immunohistochemistry applications, include appropriate negative controls (omitting primary antibody, using non-immune serum) and positive controls (tissues known to express the target). Additionally, considering the challenges in developing specific antibodies against plant proteins with high sequence homology , cross-reactivity testing with closely related proteins should be performed. RNA interference or CRISPR-Cas9 approaches to reduce target protein expression can provide further validation of antibody specificity.

What are the recommended applications for At3g15700 antibodies in plant biology research?

At3g15700 antibodies have several recommended applications in plant biology research. Primary applications include ELISA and Western blotting for detection and quantification of the target protein . For Western blotting, a dilution range of 1:500 to 1:2000 is typically effective, though optimal concentrations should be determined empirically for each experimental system. The antibody can be utilized in immunohistochemistry to localize At3g15700 protein within plant tissues, providing insights into its spatial distribution. Co-immunoprecipitation experiments can help identify protein-protein interactions involving At3g15700, elucidating its role in disease resistance pathways. Chromatin immunoprecipitation (ChIP) may be applicable if At3g15700 has DNA-binding properties or associates with chromatin-modifying complexes. For studying protein dynamics during pathogen infection, the antibody can be used in time-course experiments to monitor expression changes. Additionally, the antibody might be valuable for proximity ligation assays to detect protein interactions in situ. When designing experiments, researchers should consider the polyclonal nature of the antibody and its potential cross-reactivity with closely related proteins.

How can At3g15700 antibodies be utilized in studying plant-pathogen interactions and disease resistance mechanisms?

At3g15700 antibodies offer powerful tools for elucidating plant-pathogen interactions and disease resistance mechanisms in Arabidopsis thaliana. For investigating temporal dynamics, researchers can employ time-course studies following pathogen challenge, using the antibody to monitor At3g15700 protein accumulation or modification across multiple timepoints. Subcellular fractionation combined with immunoblotting can reveal translocation events triggered by pathogen perception. Co-immunoprecipitation with At3g15700 antibodies followed by mass spectrometry analysis can identify interaction partners that may form disease resistance complexes. For spatial distribution studies, immunohistochemistry or immunofluorescence microscopy with At3g15700 antibodies can pinpoint localization patterns during infection.

To establish functional relationships, correlate At3g15700 protein levels with disease resistance phenotypes in various genetic backgrounds. Similar approaches using fusion proteins have demonstrated effectiveness in plant protection against fungal infection . Researchers can assess post-translational modifications of At3g15700 during immune responses by combining the antibody with phospho-specific or ubiquitin-specific detection methods. For comprehensive pathway analysis, integrate antibody-based protein detection with transcriptomic and metabolomic data to construct holistic models of disease resistance networks. When experimental design permits, consider complementary strategies such as expressing recombinant antibody fragments in planta to modulate protein function, as demonstrated with Fusarium-specific antibodies linked to antifungal peptides .

What methodological considerations are important when using At3g15700 antibodies in conjunction with stress response studies?

When integrating At3g15700 antibodies into stress response studies, several methodological considerations are critical for robust data generation. First, establish comprehensive baseline expression patterns of At3g15700 protein under standard growth conditions before applying stress treatments. Design experiments with appropriate temporal resolution, as stress responses often involve rapid, transient protein dynamic changes that could be missed with limited sampling points. Consider that different stress types may induce varying patterns of At3g15700 expression or modification; previous work with catalase genes in Arabidopsis has demonstrated stress-specific responses .

For experimental controls, include both positive controls (known stress-responsive proteins) and negative controls (proteins unaffected by the stressor) alongside At3g15700 detection. When possible, complement protein-level analysis with transcriptional studies to distinguish between transcriptional and post-transcriptional regulation. Critical methodological variables include protein extraction buffers, which should be optimized to preserve potential post-translational modifications triggered by stress responses. Consider that stress conditions may alter protein complex formation; therefore, native PAGE alongside denaturing SDS-PAGE may reveal important functional information. For reproducibility, strictly standardize plant growth conditions, stress application protocols, and tissue sampling procedures to minimize experimental variability. Finally, correlate At3g15700 protein dynamics with physiological parameters and stress tolerance phenotypes to establish functional relevance, similar to approaches used in comprehensive catalase family analyses in Arabidopsis .

How can researchers address potential cross-reactivity issues when working with At3g15700 antibody in complex plant protein mixtures?

Addressing cross-reactivity issues with At3g15700 antibody requires a multi-faceted approach to ensure experimental validity. Begin with comprehensive pre-absorption controls using recombinant At3g15700 protein to confirm signal specificity. Employ genetic controls including At3g15700 knockout/knockdown lines alongside wild-type samples to distinguish specific from non-specific signals. For Western blot applications, optimize blocking conditions and antibody concentrations through systematic titration experiments to minimize background signals. Consider employing two-dimensional gel electrophoresis before immunoblotting to better resolve proteins with similar molecular weights but different isoelectric points.

When interpreting results, compare observed band patterns with predicted molecular weights of At3g15700 and potential cross-reactive proteins. For immunoprecipitation applications, incorporate stringent washing steps and validate pulled-down proteins through mass spectrometry. The challenge of obtaining specific antibodies against plant protein family members with high sequence homology is well-documented , making thorough validation essential. For critical experiments, consider using multiple antibodies targeting different epitopes of At3g15700 when available. Competitive binding assays with purified recombinant proteins can help quantify relative cross-reactivity. Additionally, pre-clearing lysates with appropriate control antibodies before target immunoprecipitation can reduce non-specific binding. Finally, incorporate epitope-tagged At3g15700 expression systems as complementary approaches when antibody cross-reactivity cannot be fully eliminated.

What are the advanced immunological techniques that can be combined with At3g15700 antibody for studying protein-protein interactions in plant immunity?

Advanced immunological techniques can significantly enhance the utility of At3g15700 antibody for investigating protein-protein interactions in plant immunity. Proximity ligation assay (PLA) combines At3g15700 antibody with antibodies against suspected interaction partners to visualize protein associations in situ with high sensitivity, providing spatial context to interactions. Förster resonance energy transfer (FRET) paired with immunofluorescence using fluorophore-conjugated At3g15700 antibody can detect close proximity between proteins in fixed samples. For dynamic interaction studies, bimolecular fluorescence complementation (BiFC) can be combined with immunoprecipitation using At3g15700 antibody to validate observed interactions.

Mass spectrometry-based immunoprecipitation approaches such as AP-MS (affinity purification-mass spectrometry) can identify novel interaction partners of At3g15700 during immune responses. Cross-linking immunoprecipitation (CLIP) techniques can capture transient interactions that might be missed by conventional co-immunoprecipitation. For studying interactions within specific cellular compartments, combine subcellular fractionation with co-immunoprecipitation using At3g15700 antibody. Super-resolution microscopy techniques like STORM or PALM with immunolabeling can provide nanoscale resolution of protein co-localization. To capture dynamic changes in interactomes during pathogen challenge, implement time-resolved interaction studies using sequential immunoprecipitations. Drawing from approaches used with other antibodies in plant research , consider expressing fusion constructs combining target domains with reporter proteins to complement antibody-based detection methods. Finally, protein arrays probed with At3g15700 antibody can screen for interactions in a high-throughput manner.

How can researchers design effective epitope mapping experiments to characterize the binding properties of At3g15700 antibody?

Designing effective epitope mapping experiments for At3g15700 antibody requires a systematic approach that employs multiple complementary methods. Begin with peptide array analysis using overlapping synthetic peptides spanning the entire 375-amino acid sequence of At3g15700 to identify primary linear epitopes recognized by the antibody. Complement this with alanine scanning mutagenesis, where individual amino acids within identified epitope regions are sequentially substituted with alanine to pinpoint critical residues for antibody binding. For conformational epitope analysis, employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of the protein protected from deuterium exchange when bound to the antibody.

X-ray crystallography or cryo-electron microscopy of antibody-antigen complexes can provide high-resolution structural insights into epitope-paratope interactions. For polyclonal antibodies like the At3g15700 antibody , epitope extraction followed by mass spectrometry can identify the collective epitope repertoire recognized by the antibody population. Competition binding assays using fragments of At3g15700 can confirm mapped epitopes and determine relative binding affinities. Surface plasmon resonance (SPR) with immobilized peptides or protein fragments can quantitatively measure binding kinetics to different epitope regions. To assess epitope conservation, perform sequence alignment analyses comparing At3g15700 with related proteins to identify unique versus conserved epitope regions. Similar strategies have been successfully employed with other antibodies, such as the structural characterization of minimal antibodies against human proteins . Finally, cross-linking mass spectrometry can provide distance constraints between antibody and antigen residues, further refining epitope mapping.

What considerations are important when designing quantitative immunoassays for measuring At3g15700 protein levels in different plant tissues and under various experimental conditions?

Designing quantitative immunoassays for At3g15700 protein requires careful consideration of multiple technical factors. First, develop a robust protein extraction protocol optimized for different plant tissues, accounting for tissue-specific differences in protein content, interfering compounds, and proteolytic activity. Include appropriate protease and phosphatase inhibitors to preserve native protein state and modifications. Establish a reliable standard curve using purified recombinant At3g15700 protein covering the expected physiological concentration range. For ELISA development, optimize antibody concentrations, blocking conditions, and detection systems through systematic titration experiments to achieve maximum sensitivity and specificity .

Validate assay linearity, precision, accuracy, and lower limit of quantification using spike-recovery experiments with recombinant protein in matrix-matched samples. Employ proper normalization strategies, such as total protein quantification or stable reference proteins, to account for sample-to-sample variations. For comparing At3g15700 levels across different experimental conditions, process all samples simultaneously when possible to minimize inter-assay variability. Investigate potential matrix effects in different tissue types that might interfere with antibody binding. Consider developing tissue-specific calibration curves if significant matrix effects are observed. Include appropriate negative controls, such as samples from At3g15700 knockout lines, to establish assay specificity. For high-throughput applications, adapt protocols to microplate formats with automated processing. Finally, verify key findings using orthogonal methods such as mass spectrometry-based protein quantification to confirm immunoassay results, especially for critical comparisons.

What future research directions could advance our understanding of At3g15700 function using antibody-based approaches?

Future research employing At3g15700 antibodies could substantially advance our understanding of this putative disease resistance protein through several promising directions. Single-cell immunolabeling techniques could reveal cell type-specific expression patterns and subcellular localization dynamics during pathogen challenge. Phospho-specific antibodies targeting predicted phosphorylation sites on At3g15700 could illuminate activation mechanisms and signal transduction pathways. Chromatin immunoprecipitation sequencing (ChIP-seq) might identify potential DNA-binding activities if At3g15700 functions as a transcriptional regulator during immune responses. Integrating antibody-based proximity labeling approaches like BioID or APEX2 with At3g15700 could map its proximal protein environment in living cells.