At1g51802 Antibody

What is At1g51802 and why is it significant in plant research?

At1g51802 encodes Defensin-like protein 166 (P0CAY5) in Arabidopsis thaliana, belonging to a class of small cysteine-rich proteins involved in plant immune responses. This protein plays a crucial role in the plant's defense mechanisms against pathogens, making it a significant target for research investigating plant immunity and stress responses. Antibodies targeting this protein enable researchers to study its expression patterns, localization, and functional role in plant defense pathways .

What applications are At1g51802 antibodies validated for?

At1g51802 antibodies are primarily validated for Western blotting (WB) with an AbInsure™ positive rating. While the primary application is WB, these antibodies may also be suitable for immunofluorescence (IF), immunohistochemistry (IHC), and immunoprecipitation (IP)/chromatin immunoprecipitation (ChIP) depending on specific validation status. Most commercially available antibodies can detect as little as 0.01-1ng of the corresponding immunogen peptide in dot blot assays, providing sufficient sensitivity for most research applications .

How should At1g51802 antibodies be stored and handled?

For optimal performance and longevity, At1g51802 antibodies are typically shipped in lyophilized form at room temperature. Upon receipt, researchers should:

• Briefly centrifuge the vial before opening

• Reconstitute by adding the recommended volume of sterile water

• Store reconstituted antibody at -20°C

• Avoid repeated freeze-thaw cycles by aliquoting into multiple tubes

• When using for experiments, keep on ice and return to storage promptly

Following these storage guidelines ensures antibody stability and consistent performance across multiple experiments .

What is the recommended starting dilution for Western blot applications?

The recommended starting dilution for At1g51802 antibodies in Western blotting applications is 1:1000. This dilution typically provides optimal signal-to-noise ratio while conserving antibody. Each antibody in these packages is capable of detecting between 0.01-1ng of its corresponding immunogen peptide in dot blot assays. Researchers should optimize the dilution based on their specific sample type, protein expression level, and detection method .

How should I design experiments to validate At1g51802 antibody specificity?

Validating antibody specificity is crucial for reliable results. A comprehensive validation approach includes:

• Positive and negative controls: Use wild-type Arabidopsis tissue (positive control) and At1g51802 knockout/knockdown lines (negative control)

• Peptide competition assay: Pre-incubate antibody with excess immunizing peptide before application

• Cross-reactivity assessment: Test against related defensin proteins

• Multiple detection methods: Compare results across Western blot, immunohistochemistry, and immunofluorescence

• Recombinant protein standard: Include purified recombinant At1g51802 protein as reference

This rigorous validation ensures that observed signals genuinely represent At1g51802 protein rather than non-specific binding .

What are the key considerations when performing immunoprecipitation with At1g51802 antibodies?

When conducting immunoprecipitation with At1g51802 antibodies, consider these critical factors:

• Sample preparation: Use fresh tissue and optimize lysis buffer composition to preserve protein-protein interactions

• Pre-clearing: Remove non-specific binding proteins using protein A/G beads before adding antibody

• Antibody amount: Typically 1-5 μg per 500 μg of total protein

• Incubation conditions: Overnight at 4°C with gentle rotation

• Washing stringency: Balance between removing non-specific interactions and preserving true interactions

• Elution method: Consider native elution with peptide competition for downstream functional assays

Following these guidelines will significantly improve the specificity and yield of your immunoprecipitation experiments .

How can I optimize Western blotting protocols specifically for At1g51802 detection?

To optimize Western blotting for At1g51802 detection:

These optimized conditions account for the specific characteristics of defensin-like proteins and maximize detection sensitivity while minimizing background .

How can I use At1g51802 antibodies to study protein-protein interactions in plant immunity pathways?

To investigate protein-protein interactions involving At1g51802:

• Co-immunoprecipitation (Co-IP): Use At1g51802 antibodies to pull down the protein complex, followed by mass spectrometry or Western blotting to identify interacting partners

• Proximity ligation assay (PLA): Combine At1g51802 antibody with antibodies against suspected interacting proteins to visualize interactions in situ

• Bimolecular fluorescence complementation (BiFC): Though not directly using antibodies, this complements antibody-based approaches

• FRET/FLIM analysis: When combined with appropriate fluorescent secondary antibodies

These approaches can reveal dynamic interaction networks during pathogen challenge or stress conditions, providing mechanistic insights into defensin functionality .

What methods can be used to quantify At1g51802 expression levels across different plant tissues or conditions?

For quantitative analysis of At1g51802 expression:

• Quantitative Western blotting: Use recombinant protein standards to create a calibration curve

• Enzyme-linked immunosorbent assay (ELISA): Develop a sandwich ELISA using At1g51802 antibodies

• Immunohistochemistry with image analysis: Quantify fluorescence intensity across different tissues

• Flow cytometry: For single-cell quantification in protoplast preparations

When comparing expression levels:

• Normalize to reliable housekeeping proteins (e.g., actin, tubulin)

• Include internal controls across blots/experiments

• Use statistical analysis to determine significance of observed differences

This multi-method approach provides robust quantification of At1g51802 expression patterns in response to various stimuli or developmental stages .

How do monoclonal and polyclonal At1g51802 antibodies compare for different research applications?

Both antibody types have distinct advantages: monoclonal antibodies offer higher specificity and reproducibility, while polyclonal antibodies often provide stronger signals by recognizing multiple epitopes. For critical experiments, validating results with both antibody types is recommended .

What are common issues when working with At1g51802 antibodies and how can they be resolved?

Addressing these common issues systematically will improve consistency and reliability of results when working with At1g51802 antibodies .

How can I distinguish between specific and non-specific binding in immunolabeling experiments?

To differentiate between specific and non-specific binding:

• Include appropriate controls:

  • No primary antibody control

  • Isotype control (irrelevant antibody of same isotype)

  • Pre-absorption with immunizing peptide

  • Tissue from knockout/knockdown plants

• Perform competitive inhibition:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare labeling pattern with and without competition

• Cross-validate with orthogonal methods:

  • Compare protein localization with GFP-fusion studies

  • Correlate with mRNA expression data

• Examine expected localization patterns:

  • Defensins typically localize to cell periphery and extracellular spaces

  • Unexpected subcellular localization may indicate non-specific binding

These approaches collectively provide confidence in the specificity of observed signals .

What analytical approaches help resolve contradictory results when using At1g51802 antibodies across different experimental systems?

When facing contradictory results:

• Systematic validation:

  • Test antibody specificity in each experimental system

  • Verify protein expression using complementary methods (RT-qPCR, mass spectrometry)

• Epitope accessibility analysis:

  • Different fixation methods may mask epitopes

  • Test multiple antibodies targeting different regions of At1g51802

• Consider biological variables:

  • Plant age, growth conditions, and stress exposure affect protein expression

  • Different ecotypes may have slight sequence variations affecting antibody binding

• Statistical approach:

  • Increase biological replicates

  • Apply appropriate statistical tests to determine significance of observed differences

  • Perform meta-analysis across experiments

• Detailed method documentation:

  • Maintain comprehensive records of all experimental conditions

  • Consider creating a standardized protocol for community use

This systematic analytical framework helps resolve apparent contradictions and advances understanding of At1g51802 function .

How can I develop custom At1g51802 antibodies for specialized research applications?

Developing custom At1g51802 antibodies involves several key steps:

• Epitope selection:

  • Analyze protein sequence for immunogenic regions

  • Avoid regions with high homology to other defensins

  • Consider synthesizing peptides corresponding to N-terminal or unique domains

• Immunization strategy:

  • Select appropriate animal host (typically rabbit, mouse, or rat)

  • Design immunization schedule with primary and booster injections

  • Monitor antibody titers through ELISA

• For monoclonal antibody production:

  • Isolate B lymphocytes from immunized mice

  • Fuse with myeloma cells to create hybridomas

  • Screen hybridoma clones for antibody production

  • Expand positive clones for antibody harvesting

• Purification methods:

  • Protein A/G affinity chromatography for whole IgG

  • Antigen-specific affinity purification for highest specificity

• Validation requirements:

  • Test against recombinant protein

  • Verify specificity in wild-type vs. knockout samples

  • Cross-reactivity assessment

Custom antibody development typically requires 3-4 months and specialized facilities, but yields reagents perfectly tailored to specific research needs .

What are the considerations for combining At1g51802 antibodies with other techniques like CRISPR-Cas9 genome editing?

Integrating At1g51802 antibodies with genome editing approaches:

• Epitope tagging strategies:

  • Use CRISPR-Cas9 to add epitope tags (HA, FLAG, etc.) to endogenous At1g51802

  • Compare protein detection between At1g51802-specific antibodies and anti-tag antibodies

  • Consider tag position effects on protein function

• Knockout validation:

  • Generate CRISPR-Cas9 knockouts of At1g51802

  • Use antibodies to confirm protein absence

  • Quantify knockout efficiency in heterogeneous populations

• Domain function analysis:

  • Create domain-specific deletions or modifications

  • Use domain-specific antibodies to track expression and localization

  • Correlate structural changes with functional outcomes

• Complementation studies:

  • Reintroduce modified At1g51802 variants into knockout lines

  • Use antibodies to verify expression levels

  • Normalize phenotypic rescue to protein expression levels

This integrated approach leverages the strengths of both antibody-based detection and precise genome manipulation to gain deeper insights into At1g51802 function .

How can paired antibody sequencing data improve At1g51802 antibody development and specificity?

Recent advances in antibody sequencing can enhance At1g51802 antibody research:

• Sequence-based optimization:

  • Analyze paired heavy/light chain sequences of effective antibodies

  • Identify complementarity-determining regions (CDRs) crucial for At1g51802 binding

  • Engineer improved variants with enhanced specificity or affinity

• Germline analysis benefits:

  • Examine germline gene usage patterns in successful At1g51802 antibodies

  • Select optimal framework regions for humanization or stabilization

  • Predict cross-reactivity based on germline sequence conservation

• Application to phage display:

  • Create synthetic antibody libraries based on successful sequence patterns

  • Screen libraries against different At1g51802 epitopes

  • Rapid development of application-specific variants

• Bioinformatic integration:

  • Compare antibody sequences with epitope structures

  • Predict binding interfaces through computational modeling

  • Design rational mutations to enhance performance

The PairedAbNGS database and similar resources provide valuable reference data for these approaches, potentially yielding next-generation At1g51802 antibodies with superior characteristics .

What are the future directions for At1g51802 antibody research in plant immunology?

The future of At1g51802 antibody research in plant immunology will likely focus on several emerging areas:

These directions will continue to expand our understanding of plant defensin biology and potentially contribute to agricultural innovation .

How should researchers document and publish At1g51802 antibody validation for reproducible science?

To ensure reproducibility in At1g51802 antibody research, documentation should include:

• Comprehensive antibody information:

  • Catalog number and lot number

  • Host species and antibody type (monoclonal/polyclonal)

  • Immunogen sequence and location within the protein

  • Validation methods and results

• Detailed experimental protocols:

  • Complete methods for sample preparation

  • Antibody dilutions and incubation conditions

  • Buffer compositions and washing procedures

  • Image acquisition parameters

• Control experiments:

  • All controls used to verify specificity

  • Representative images of positive and negative controls

  • Quantification methods and statistical analyses

• Research resource identifiers (RRIDs):

  • Include antibody RRIDs in publications

  • Submit validation data to antibody validation databases

  • Consider depositing protocols in repositories like protocols.io