At2g20463 Antibody

What is the At2g20463 protein in Arabidopsis thaliana?

At2g20463 (UniProt accession: Q2V476) is a protein expressed in Arabidopsis thaliana (Mouse-ear cress) . Based on gene annotation and sequence analysis, At2g20463 belongs to a family of plant proteins that may be involved in defense mechanisms. The gene is located on chromosome 2 of Arabidopsis thaliana and may share characteristics with defensin-like proteins, which are important components of plant innate immunity . Defensins in Arabidopsis have evolved through various mechanisms including natural selection and recombination, though they show a predominantly neutral evolutionary pattern compared to NLR proteins which exhibit more frequent positive selection .

What applications are supported for At2g20463 Antibody?

At2g20463 Antibody has been validated for the following laboratory applications:

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Western Blotting (WB)

The antibody is specifically designed for research identification of the antigen and should not be used for diagnostic or therapeutic procedures . When designing experiments, researchers should consider that this antibody is polyclonal in nature, which may provide broader epitope recognition but potentially more batch-to-batch variation compared to monoclonal antibodies.

What are the key specifications of commercially available At2g20463 Antibody?

These specifications are important when planning experimental timelines and designing appropriate control experiments .

What are the optimal conditions for using At2g20463 Antibody in Western blotting?

For optimal Western blot results with At2g20463 Antibody, follow these methodological guidelines:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing protease inhibitors

  • Load 10-30 μg of total protein per lane

  • Include both positive controls (recombinant At2g20463) and negative controls

• Electrophoresis and transfer:

  • Use 12-15% SDS-PAGE gels for optimal resolution of lower molecular weight defensin-like proteins

  • Transfer to PVDF membrane (preferred over nitrocellulose for small proteins)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary antibody (At2g20463 Antibody) at 1:500 to 1:2000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST

  • Incubate with anti-rabbit HRP-conjugated secondary antibody at 1:5000-1:10000 for 1 hour at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) substrate

  • Optimal exposure times typically range from 30 seconds to 5 minutes

Similar to other antibodies used in plant research, optimization may be necessary for specific tissue types and experimental conditions .

How should samples be prepared for At2g20463 Antibody detection in ELISA?

For effective ELISA detection using At2g20463 Antibody, sample preparation is crucial:

• Plant tissue extraction:

  • Homogenize fresh or frozen Arabidopsis tissue in extraction buffer (PBS pH 7.4 with 0.05% Tween-20 and protease inhibitors)

  • Centrifuge at 12,000 × g for 15 minutes at 4°C

  • Collect supernatant for analysis

• ELISA protocol optimization:

  • Coating: Use purified recombinant At2g20463 protein (1-5 μg/ml) in carbonate buffer (pH 9.6) for standard curve

  • Blocking: 2-3% BSA in PBS for 1-2 hours at room temperature

  • Primary antibody: Dilute At2g20463 Antibody 1:1000 to 1:5000 in blocking buffer

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000 to 1:10000

  • Substrate: TMB solution for colorimetric detection

  • Reading: Measure absorbance at 450 nm

• Statistical considerations:

  • Run all samples in triplicate

  • Include standard curves using recombinant protein in each plate

  • Use appropriate negative controls (samples from knockout lines if available)

This methodological approach allows for quantitative assessment of At2g20463 protein levels in plant tissues and can be modified for competitive ELISA applications if needed .

What controls should be used when working with At2g20463 Antibody?

Proper controls are essential for antibody-based experiments to ensure reliable and interpretable results:

• Positive controls:

  • Recombinant At2g20463 protein (the immunogen used to generate the antibody)

  • Samples with known expression of At2g20463 (e.g., specific tissues or conditions where expression has been confirmed)

• Negative controls:

  • Isotype control (non-specific rabbit IgG at the same concentration)

  • Samples from At2g20463 knockout or knockdown plants

  • Secondary antibody-only controls to assess non-specific binding

  • Pre-adsorption control (antibody pre-incubated with excess antigen)

• Additional validation controls:

  • Correlation with mRNA expression data

  • Peptide competition assay

  • Testing antibody specificity against closely related proteins in the same defensin family

These controls help distinguish specific signal from background and validate antibody specificity, which is particularly important for polyclonal antibodies that may have batch-to-batch variation .

Why might At2g20463 Antibody show weak or no signal in Western blot?

Several methodological and biological factors can contribute to weak or absent signals:

Defensin-like proteins often show tissue-specific expression patterns and may be induced under specific stress conditions. If expression data indicates At2g20463 is primarily expressed in specific tissues (like pistils, as is common for defensin-like proteins in Arabidopsis), using those tissues may significantly improve detection .

How can cross-reactivity issues with At2g20463 Antibody be addressed?

Cross-reactivity can complicate interpretation of results, especially with polyclonal antibodies against members of protein families:

• Assessing potential cross-reactivity:

  • Perform sequence alignment of At2g20463 with related defensin family members

  • Test antibody against recombinant related proteins if available

  • Compare band patterns with predicted molecular weights of potential cross-reactive proteins

• Mitigation strategies:

  • Increase washing stringency (higher salt concentration, longer wash times)

  • Pre-adsorb antibody with recombinant related proteins

  • Use gradient gels with higher resolution to separate closely related proteins

  • Consider immunoprecipitation followed by mass spectrometry to confirm identity of detected proteins

• Analysis approaches:

  • Use knockout/knockdown lines of At2g20463 as negative controls

  • Employ orthogonal techniques (qRT-PCR, MS) to validate findings

  • Consider developing more specific detection methods if cross-reactivity cannot be eliminated

Understanding the evolutionary relationship between defensin family members in Arabidopsis can help predict potential cross-reactivity, as these proteins show varying degrees of conservation and selective pressures .

How can antibody validation be performed for At2g20463 Antibody?

Comprehensive validation ensures reliable experimental results and should include:

• Primary validation techniques:

  • Western blot against recombinant At2g20463 protein

  • Testing in knockout/knockdown lines (signal should be reduced/absent)

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assay

• Secondary validation approaches:

  • Correlation with mRNA expression patterns across tissues

  • Comparison with tagged-protein expression (e.g., GFP-fusion)

  • Cross-platform validation using different antibody-based techniques

• Documentation and reporting:

  • Record all validation experiments with detailed methods

  • Document antibody lot number, dilution, and conditions used

  • Report both positive and negative validation results

  • Consider sharing validation data through antibody validation repositories

This multi-faceted approach aligns with current best practices in antibody validation for research applications and helps ensure reproducibility across experiments .

How can At2g20463 Antibody be used to study plant immune responses?

At2g20463 Antibody can be a valuable tool for investigating plant immunity, particularly if the protein functions as a defensin-like molecule:

• Expression analysis during pathogen challenge:

  • Monitor At2g20463 protein levels in response to diverse pathogens (bacterial, fungal, viral)

  • Compare protein expression with transcriptional responses

  • Analyze tissue-specific induction patterns using immunohistochemistry

• Localization studies:

  • Use immunofluorescence to determine subcellular localization before and after infection

  • Investigate if localization changes during different infection phases

  • Compare with other defensin family members to identify functional similarities/differences

• Functional studies:

  • Correlate protein levels with resistance phenotypes

  • Combine with genetic approaches (overexpression, knockout lines)

  • Study protein-protein interactions during immune responses

Defensin-like proteins in Arabidopsis show varied patterns of diversification and selection compared to NLR immune receptors. Unlike NLRs which frequently undergo positive selection, defensins show predominantly neutral evolutionary patterns with infrequent positive selection events, suggesting different evolutionary constraints despite their roles in immunity .

Can At2g20463 Antibody detect post-translational modifications of the target protein?

Detection of post-translational modifications (PTMs) requires careful experimental design:

• Potential PTMs in plant defensin-like proteins:

  • Disulfide bond formation (critical for defensin structure and function)

  • Glycosylation

  • Phosphorylation

  • Proteolytic processing

• Methodological approaches:

  • Compare migration patterns under reducing vs. non-reducing conditions

  • Use phosphatase treatment to identify phosphorylated forms

  • Employ glycosidase treatments to detect glycosylated variants

  • Combine with mass spectrometry for definitive PTM mapping

• Analytical considerations:

  • Higher resolution gels may be needed to separate modified forms

  • Consider 2D gel electrophoresis to separate based on both mass and charge

  • Use specific PTM antibodies in conjunction with At2g20463 Antibody

Understanding potential PTMs is particularly relevant for secreted proteins like defensins, where processing and modification often occur during secretion and can be essential for biological activity .

What approaches can be used to study protein-protein interactions involving At2g20463?

Investigating interaction partners can provide insights into At2g20463 function:

• Co-immunoprecipitation (Co-IP) with At2g20463 Antibody:

  • Cross-link proteins in vivo before extraction

  • Immunoprecipitate with At2g20463 Antibody

  • Identify co-precipitated proteins by mass spectrometry

  • Verify interactions with reciprocal Co-IP

• Proximity-based methods:

  • Combine with BioID or TurboID proximity labeling

  • Use split complementation assays (BiFC) with tagged constructs

  • Perform FRET/FLIM analysis for direct interaction studies

• In vitro validation:

  • Express recombinant proteins for pull-down assays

  • Use surface plasmon resonance to measure binding kinetics

  • Employ yeast two-hybrid screening for systematic interaction discovery

• Functional validation:

  • Assess phenotypic consequences when interactions are disrupted

  • Investigate co-localization during defense responses

  • Study regulatory relationships among interacting partners

These approaches can help position At2g20463 within protein interaction networks related to plant immunity and stress responses, providing context for its biological function .

How does At2g20463 expression compare with other defensin family members in Arabidopsis?

Understanding the expression patterns within the defensin family provides valuable context:

• Comparative expression analysis:

  • Use At2g20463 Antibody alongside antibodies against other defensin family members

  • Compare protein expression with transcriptomic data from public databases

  • Analyze tissue specificity and developmental regulation

• Response to biotic and abiotic stresses:

  • Monitor differential expression during pathogen infection

  • Compare responses to different classes of pathogens

  • Analyze expression changes under abiotic stress conditions

• Evolutionarily informed analysis:

  • Group expression patterns according to evolutionary relationships

  • Assess if conserved defensins show similar or divergent expression patterns

  • Correlate expression patterns with evolutionary selection pressures

Research on Arabidopsis defensins indicates that many members are predominantly expressed in pistils, in contrast to NLR immune receptors which show broader expression patterns across tissues. This tissue-specific expression suggests specialized roles in reproductive tissues that may extend beyond pathogen defense .

Can At2g20463 Antibody be adapted for flow cytometric analysis of plant cells?

While not a standard application for plant proteins, flow cytometry can be adapted for certain plant research questions:

• Sample preparation for protoplasts:

  • Isolate protoplasts from Arabidopsis tissues using appropriate enzymes

  • Fix with 2-4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100 for intracellular targets

  • Block with 3% BSA in PBS

• Antibody staining protocol:

  • Primary staining: At2g20463 Antibody (1:100-1:500) for 1 hour at RT

  • Wash steps: 3× with PBS

  • Secondary staining: Fluorophore-conjugated anti-rabbit antibody (1:1000)

  • Counterstain nuclei with DAPI if needed

• Controls and validation:

  • Isotype control (rabbit IgG)

  • Secondary antibody only

  • Protoplasts from knockout lines

This methodology adapts traditional flow cytometry approaches developed for mammalian cells to plant systems, allowing quantitative analysis of protein expression at the single-cell level .