At3g16895 Antibody

What is At3g16895 and why is it significant in Arabidopsis research?

At3g16895 encodes a defensin-like (DEFL) family protein in Arabidopsis thaliana, also known as defensin-like protein 47 or cysteine-rich protein . Defensins are small cysteine-rich peptides that play crucial roles in plant innate immunity against microbial pathogens and are increasingly recognized for their roles in plant development and stress responses. The study of At3g16895 contributes to our understanding of plant defense mechanisms and potentially novel antimicrobial compounds. Research into this protein has implications for agricultural applications, particularly in developing disease-resistant crop varieties. The gene has been identified in chromatin immunoprecipitation (ChIP) studies, suggesting it may be regulated by key cell cycle control factors like Retinoblastoma-Related protein (RBR1) .

What are the key applications for At3g16895 antibody in experimental protocols?

At3g16895 antibodies are primarily used for detecting and quantifying the target protein in various experimental applications. The commercially available rabbit anti-Arabidopsis thaliana At3g16895 polyclonal antibody has been validated for applications including Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blotting (WB) . These applications allow researchers to determine protein expression levels, localization patterns, and potential interactions with other biomolecules. When using these antibodies, researchers should follow manufacturer protocols for optimal results, typically employing dilution factors ranging from 1:50 to 1:200 depending on the specific application and experimental conditions . The antibody enables investigations into the developmental expression patterns of this defensin-like protein and its potential roles in plant immunity and stress responses.

How should At3g16895 antibody be stored and handled for maximum efficacy?

Proper storage and handling of antibodies is critical for maintaining their activity and specificity. For At3g16895 polyclonal antibody, follow these evidence-based practices: Store freeze-dried antibody at 2-8°C until ready for use . When rehydrating the antibody, use the specified volume of distilled water as indicated on the product specification sheet, then centrifuge if the solution appears cloudy. After rehydration, store at 2-8°C and never freeze, as freezing-thawing cycles can denature antibodies and compromise binding capacity . Prepare working dilutions on the day of use rather than storing diluted antibody for extended periods. The typical shelf life after rehydration is approximately six months, though this may be extended if quality control tests indicate acceptable performance for your specific application . Always minimize exposure to light, particularly for fluorophore-conjugated antibodies, and avoid repeated freeze-thaw cycles that can degrade antibody quality.

How can I optimize protein extraction protocols for At3g16895 detection in plant tissues?

Effective protein extraction is fundamental to successful antibody-based detection of At3g16895. For Arabidopsis tissues, a modified extraction protocol optimized for cysteine-rich proteins is recommended. Begin with fresh or flash-frozen plant material ground to a fine powder in liquid nitrogen. Use a extraction buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and freshly added protease inhibitors. For defensin-like proteins such as At3g16895, include 5 mM dithiothreitol (DTT) to maintain reduced disulfide bonds. After extraction, centrifuge at 14,000 × g for 15 minutes at 4°C and collect the supernatant. Protein concentration should be determined using Bradford or BCA assays before proceeding to immunodetection techniques. For optimal results, avoid using protein extraction buffers with high concentrations of SDS, as this may interfere with antibody-antigen interactions in certain applications like ELISA . Testing multiple extraction buffers with different detergent compositions may be necessary to determine optimal conditions for your specific plant tissue and developmental stage.

What are the critical parameters for Western blot optimization with At3g16895 antibody?

Optimizing Western blot protocols for At3g16895 detection requires careful attention to several critical parameters. First, protein separation should be performed using 15-18% SDS-PAGE gels due to the small size of defensin-like proteins (typically 5-10 kDa). Transfer conditions should be optimized for small proteins: use PVDF membranes with 0.2 μm pore size rather than 0.45 μm, and transfer at lower voltage (30V) for longer duration (2 hours) to prevent small proteins from passing through the membrane. For blocking, 5% non-fat dry milk in TBST is generally effective, but BSA-based blocking buffers may provide lower background in some cases. The optimal antibody dilution for At3g16895 detection typically falls within the 1:50 to 1:200 range as recommended by manufacturers , but this should be empirically determined. Include appropriate positive controls (recombinant At3g16895 protein if available) and negative controls (samples from knockout plants). For enhanced chemiluminescence detection, longer exposure times may be necessary due to the potentially low expression levels of defensin-like proteins. Consider using signal enhancers or more sensitive detection systems if standard protocols yield weak signals.

What controls should be included in immunoprecipitation experiments using At3g16895 antibody?

Robust controls are essential for reliable immunoprecipitation (IP) experiments with At3g16895 antibody. Include the following controls in your experimental design: (1) Input control - analyze a small portion (5-10%) of the pre-cleared lysate before IP to confirm the presence of target protein; (2) Negative antibody control - perform parallel IP with non-specific IgG from the same species as the At3g16895 antibody (rabbit IgG) ; (3) No-antibody control - conduct the entire IP procedure without adding any antibody; (4) Competitive peptide control - pre-incubate the antibody with excess purified At3g16895 protein or antigenic peptide before IP to demonstrate binding specificity. For ChIP experiments studying potential regulatory interactions with At3g16895, additional controls should include IPs from tissues where the gene is not expressed and, ideally, from At3g16895 knockout plants to establish background signal levels . When analyzing results, quantify the enrichment of your target over the IgG control using densitometry for Western blot detection or qPCR for ChIP applications. Document any non-specific bands observed and investigate potential cross-reactivity with related defensin-like proteins.

How should different fixation methods be evaluated for immunohistochemical detection of At3g16895?

When performing immunohistochemistry to localize At3g16895 protein in plant tissues, the choice of fixation method significantly impacts antibody binding and signal specificity. Compare multiple fixation protocols to determine optimal conditions: (1) Paraformaldehyde fixation (4% in PBS) preserves protein antigenicity but may provide limited tissue penetration; (2) Methanol:acetic acid fixation (3:1) offers better penetration but can denature some epitopes; (3) Glutaraldehyde fixation (0.1-2.5%) provides excellent ultrastructural preservation but often requires antigen retrieval steps. For plant tissues, additional considerations include cell wall permeability—enzymatic digestion with cellulase and macerozyme may be necessary before antibody incubation. When evaluating fixation methods, assess both signal intensity and background levels. Perform parallel staining on tissues known to express high levels of At3g16895 (positive control) and on tissues from knockout plants or developmental stages with minimal expression (negative control). Document fixation artifacts and optimize antigen retrieval methods if needed. The best fixation protocol balances tissue morphology preservation with epitope accessibility for the At3g16895 antibody .

How should researchers interpret unexpected band patterns in Western blots using At3g16895 antibody?

When encountering unexpected band patterns in Western blots with At3g16895 antibody, systematic analysis is essential. The predicted molecular weight of At3g16895 (defensin-like protein 47) is approximately 5-10 kDa, typical for defensin family proteins. Multiple bands may indicate: (1) Post-translational modifications such as glycosylation, phosphorylation, or proteolytic processing that alter molecular weight; (2) Protein dimers or multimers if sample reduction was incomplete; (3) Cross-reactivity with related defensin-like proteins, as Arabidopsis contains numerous DEFL family members with structural similarities; (4) Alternative splice variants, though less common with small defensin genes. To systematically address unexpected bands, run parallel samples with different reducing conditions, perform peptide competition assays to determine specific vs. non-specific binding, and compare expression patterns in different tissues and developmental stages. For definitive identification of unexpected bands, consider mass spectrometry analysis of the excised gel bands. Record all observations methodically, as unexpected bands may actually reveal novel biological phenomena such as previously undocumented protein modifications or interactions relevant to At3g16895 function .

How can researchers validate the specificity of At3g16895 antibody in their experimental systems?

Validating antibody specificity is critical for generating reliable data with At3g16895 antibody. Implement these complementary validation strategies: (1) Genetic validation - compare antibody signals between wild-type plants and At3g16895 knockout/knockdown lines, where the specific signal should be absent or significantly reduced; (2) Peptide competition assay - pre-incubate the antibody with purified antigen peptide before application to samples, which should abolish specific binding; (3) Orthogonal detection methods - correlate antibody-based detection with mRNA expression using RT-qPCR or RNA-seq data for the same tissues/conditions; (4) Cross-platform validation - confirm consistent results across different techniques (e.g., Western blot, immunofluorescence, ELISA) . For advanced validation, consider using epitope-tagged At3g16895 expressed in Arabidopsis under native promoter control, then compare detection patterns between the antibody against the native protein and an antibody against the epitope tag. Document all validation experiments thoroughly, including positive and negative controls, antibody dilutions, and detection parameters. Comprehensive validation not only ensures experimental reliability but also contributes to research reproducibility in the broader scientific community.

What approaches help distinguish between specific signal and background in immunofluorescence studies of At3g16895?

Distinguishing specific signal from background is particularly challenging when studying low-abundance proteins like At3g16895. Implement these methodological approaches to maximize signal-to-noise ratio: (1) Optimized blocking - test different blocking agents (BSA, normal serum, commercial blockers) to minimize non-specific binding; (2) Adsorption controls - pre-adsorb the antibody with plant tissue powder from At3g16895 knockout plants to remove antibodies with off-target affinities; (3) Secondary antibody controls - include samples treated with secondary antibody only to identify non-specific binding; (4) Autofluorescence controls - capture images of unstained tissue to identify natural plant autofluorescence, particularly from chlorophyll and cell wall components, which can be subtracted during image analysis . When using fluorescent markers like APC (allophycocyanin), select excitation and emission settings that minimize overlap with plant autofluorescence . For multi-channel imaging, perform proper channel compensation based on single-stained controls. Consider advanced techniques like spectral unmixing to separate overlapping fluorescence signals. Always standardize image acquisition parameters (exposure time, gain) across experimental and control samples, and process all images using identical adjustments for accurate comparative analysis.

How can At3g16895 antibody be integrated into ChIP-seq workflows to study transcriptional regulation?

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) using At3g16895 antibody requires careful optimization for studying protein-DNA interactions. Based on the identification of At3g16895 in RBR1 ChIP studies , researchers may want to investigate how this defensin-like gene is regulated at the chromatin level. For successful ChIP-seq experiments, first validate the At3g16895 antibody specifically for ChIP applications using ChIP-qPCR on known target regions. Optimize crosslinking conditions (typically 1% formaldehyde for 10-15 minutes) for plant tissues, considering that excessive crosslinking can mask epitopes. For chromatin fragmentation, sonication parameters should be calibrated to achieve fragments of 200-500 bp. The immunoprecipitation step requires optimized antibody concentrations, typically 2-5 μg per reaction, with parallel IgG control IPs . For sequencing library preparation, use methods optimized for low-input samples if the target protein has limited abundance or narrowly defined expression patterns. During data analysis, employ peak calling algorithms appropriate for transcription factors (e.g., MACS2) or histone modifications (e.g., SICER) depending on your research question. Validate ChIP-seq findings with orthogonal methods such as reporter gene assays or EMSA to confirm functional significance of identified binding sites.

What approaches enable the study of post-translational modifications of At3g16895 protein?

Investigating post-translational modifications (PTMs) of At3g16895 requires integration of immunological and mass spectrometry techniques. Begin by immunoprecipitating the native protein from plant tissues using the At3g16895 antibody under non-denaturing conditions to preserve modifications . For phosphorylation studies, supplement lysis buffers with phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate). After immunoprecipitation, separate proteins by SDS-PAGE and perform parallel Western blots with the At3g16895 antibody and modification-specific antibodies (anti-phospho, anti-ubiquitin, etc.). For comprehensive PTM mapping, excise gel bands containing At3g16895 for mass spectrometry analysis. Use both collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation methods, as they provide complementary information for different PTM types. Implement targeted mass spectrometry approaches like parallel reaction monitoring (PRM) or multiple reaction monitoring (MRM) for higher sensitivity when studying low-abundance modifications. To study PTM dynamics during development or stress responses, perform time-course experiments with appropriate biological replicates. Functional significance of identified PTMs can be further investigated using site-directed mutagenesis of modified residues followed by functional assays in transgenic plants.

How can multimodal data integration enhance At3g16895 protein characterization studies?

Multimodal data integration provides a comprehensive understanding of At3g16895 function by combining multiple experimental approaches. Similar to the CBMC analysis described in the search results , researchers can implement integrated analysis workflows that combine protein-level data (from antibody-based detection) with transcriptomic data. Begin by collecting paired datasets: (1) Protein expression data using At3g16895 antibody in techniques like Western blot, immunofluorescence, or ELISA; (2) Transcript expression data using RT-qPCR or RNA-seq; (3) Protein interaction data from co-immunoprecipitation followed by mass spectrometry. For data integration, normalize each dataset appropriately and use correlation analyses to identify relationships between protein abundance, mRNA levels, and interacting partners across tissues or conditions. Visualization tools like FeatureScatter plots can reveal relationships between protein and RNA measurements . Consider implementing multimodal computational approaches such as MOFA (Multi-Omics Factor Analysis) or DIABLO (Data Integration Analysis for Biomarker discovery using Latent cOmponents) to identify patterns across datasets. This integrated approach can reveal post-transcriptional regulation mechanisms affecting At3g16895, identify functional protein complexes, and characterize regulatory networks controlling defensin expression in response to environmental stimuli or developmental cues.

What experimental designs can reveal At3g16895 function during plant immune responses?

To investigate At3g16895 function in plant immunity, implement comprehensive experimental designs that exploit the specificity of the At3g16895 antibody. Design time-course experiments where Arabidopsis plants are challenged with different pathogens (bacterial, fungal, oomycete) followed by protein extraction and immunoblotting with At3g16895 antibody at defined time points (0, 3, 6, 12, 24, 48 hours post-infection) . Complement protein expression data with subcellular localization studies using immunogold electron microscopy to determine if At3g16895 accumulates at infection sites or undergoes relocalization during immune responses. For functional studies, generate transgenic Arabidopsis lines with modulated At3g16895 expression (overexpression, RNAi knockdown, CRISPR knockout) and assess disease susceptibility using quantitative pathogen growth assays. Compare protein expression patterns across these genetically modified lines using the antibody. Investigate potential antimicrobial activity by expressing and purifying recombinant At3g16895 protein (detected and validated using the antibody) for in vitro pathogen growth inhibition assays. For systems-level analysis, perform immunoprecipitation coupled with mass spectrometry to identify interaction partners during immune responses, potentially revealing mechanistic insights into At3g16895's role in plant defense signaling networks .

What are the optimal experimental conditions for At3g16895 antibody applications?

This comprehensive comparison of experimental conditions provides researchers with a starting point for protocol optimization when working with At3g16895 antibody . These parameters should be empirically validated in each laboratory's specific experimental system, as optimal conditions may vary based on tissue type, developmental stage, and experimental equipment.

How does At3g16895 expression compare with related defensin-like proteins across plant tissues?

This comparative expression table synthesizes potential patterns based on known defensin-like protein behaviors, highlighting the tissue-specific expression patterns that might be expected when using At3g16895 antibody . The inclusion of RBR1 as a potential regulatory factor reflects the identification of At3g16895 in RBR1 ChIP studies, suggesting cell cycle-related regulation may influence expression patterns.