At1g63350 Antibody

What is AT1G63350 and why is it significant in plant immunity research?

AT1G63350 encodes a disease resistance protein belonging to the CC-NBS-LRR (Coiled-Coil-Nucleotide-Binding Site-Leucine-Rich Repeat) class in Arabidopsis thaliana. This protein functions in ATP binding and is involved in defense responses and N-terminal protein myristoylation according to computational analysis . The protein contains characteristic domains including leucine-rich repeats and the NB-ARC domain, which are hallmarks of plant resistance (R) proteins.

The significance of AT1G63350 and similar R-genes lies in their crucial role in plant immunity. Studies have demonstrated that expression levels of R-genes directly correlate with resistance to specific pathogens. Most investigations have found that increased expression of R-genes leads to significant decreases in pathogen load, highlighting their importance in plant defense mechanisms . AT1G63350 represents an important model for understanding how plants recognize and respond to pathogen invasion, making it a valuable target for research on plant immunity pathways.

R-genes like AT1G63350 are particularly interesting because their expression and function may vary depending on environmental conditions. Research has identified interactions between R-genes, climate variables, and pathogen load, suggesting that selection may favor different levels of R-gene expression in different environments . This ecological dimension adds another layer of significance to AT1G63350 research, potentially informing agricultural strategies for disease resistance.

What approaches are recommended for generating specific antibodies against AT1G63350?

Generating specific antibodies against AT1G63350 requires careful consideration of epitope selection and antibody format. Based on successful approaches with other proteins, researchers should consider the following strategies:

• Epitope selection: For optimal specificity, target unique regions of AT1G63350 that have minimal sequence homology with related CC-NBS-LRR proteins. This approach is particularly important given that plant genomes often contain multiple related R-genes. Epitopes can be selected from either:

  • The N-terminal coiled-coil domain, which typically shows greater sequence diversity among R-proteins

  • Specific segments of the leucine-rich repeat region that are unique to AT1G63350

  • The NB-ARC domain, if containing distinguishing sequences

• Antibody format options:

  • Monoclonal antibodies offer high specificity and reproducibility, similar to those developed for AT1 receptors

  • Polyclonal antibodies may provide broader epitope recognition but require careful validation

  • Single-domain antibodies can be engineered for enhanced avidity through multimerization, as demonstrated with other proteins

Researchers have successfully generated monoclonal antibodies against other proteins by immunizing mice with synthetic peptides representing specific sequences from extracellular or intracellular domains . This approach could be adapted for AT1G63350, focusing on unique peptide sequences identified through computational analysis.

It is advisable to develop antibodies against multiple epitopes of AT1G63350 to ensure robust detection and to provide internal validation. This multi-epitope approach has been successfully employed for other proteins and can help mitigate the risk of epitope masking due to protein interactions or conformational changes.

How can researchers validate the specificity of AT1G63350 antibodies?

Validating antibody specificity is crucial, especially given documented issues with commercial antibodies leading to protein misidentification . For AT1G63350 antibodies, a comprehensive validation approach should include:

• Western blot analysis using multiple controls:

  • Wild-type Arabidopsis thaliana samples (positive control)

  • AT1G63350 knockout or knockdown lines (negative control)

  • Recombinant AT1G63350 protein (positive control)

  • Closely related CC-NBS-LRR proteins to assess cross-reactivity

  The expected molecular weight of AT1G63350 is approximately 102.6 kDa based on the protein sequence , which should be used as a reference point when evaluating band specificity.

• Immunoprecipitation followed by mass spectrometry:

  • This approach can confirm that the antibody is capturing the intended target

  • Analysis of co-immunoprecipitated proteins can identify potential cross-reactive proteins

• Immunohistochemistry with appropriate controls:

  • Compare staining patterns in tissues known to express AT1G63350

  • Include knockout lines as negative controls

  • Use competitive blocking with the immunizing peptide to confirm specificity

• Dot blot analysis with synthetic peptides:

  • Test antibody binding against the immunizing peptide

  • Include closely related peptide sequences to assess cross-reactivity

Specificity validation is particularly important for AT1G63350 because it shares significant sequence homology with other disease resistance proteins. For instance, AT1G63350 has high sequence similarity with AT1G62630.1, according to computational analysis . This homology increases the risk of cross-reactivity, necessitating rigorous validation protocols.

What are the optimal sample preparation methods for AT1G63350 antibody applications?

Sample preparation is critical for successful detection of AT1G63350 using antibodies. The following methods are recommended based on successful protocols for similar plant proteins:

• Protein extraction from plant tissues:

  • Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail

  • Include phosphatase inhibitors if studying phosphorylation states

  • Consider adding N-ethylmaleimide to preserve potential ubiquitination or other thiol-dependent modifications

  • Optimize extraction conditions through temperature control (4°C is typically recommended)

• Sample handling for immunoblotting:

  • Avoid repeated freeze-thaw cycles of protein extracts

  • Optimize protein loading (typically 20-50 μg of total protein)

  • Consider using gradient gels (4-12%) for optimal resolution of AT1G63350

  • Include a reducing agent in sample buffer to ensure consistent protein conformation

• Tissue preparation for immunohistochemistry:

  • Fix tissues in 4% paraformaldehyde for 2-4 hours at 4°C

  • Consider antigen retrieval methods to expose epitopes that may be masked during fixation

  • Optimize blocking conditions to minimize background signal

  • Include permeabilization steps for antibodies targeting intracellular domains

• Considerations for different plant tissues:

  • AT1G63350 has been reported to be expressed in multiple plant structures

  • Expression may vary depending on developmental stage and environmental conditions

  • Sample collection timing should be optimized based on expected expression patterns

  • Consider tissue-specific extraction protocols for challenging tissues

For optimal results, researchers should adjust these protocols based on the specific properties of their AT1G63350 antibody and the plant material being studied. Preliminary experiments comparing different extraction and preparation methods are recommended to establish optimal conditions.

How can researchers overcome cross-reactivity issues with AT1G63350 antibodies?

Cross-reactivity is a significant challenge when working with antibodies against plant R-proteins like AT1G63350, primarily due to sequence conservation among related proteins. To address this issue, researchers can implement several advanced strategies:

• Epitope refinement through computational analysis:

  • Perform comprehensive sequence alignment of AT1G63350 with related R-proteins

  • Identify unique sequences with minimal homology to related proteins

  • Use structural prediction tools to select surface-exposed regions that maintain native conformation

  • Prioritize regions with distinctive post-translational modifications

• Antibody purification techniques:

  • Implement affinity purification using the immunizing peptide

  • Apply negative selection against peptides from closely related proteins

  • Consider cross-adsorption against tissue lysates from AT1G63350 knockout lines

  • Use sequential affinity purification to enhance specificity

• Validation in multiple experimental systems:

  • Compare antibody performance in both native and denatured conditions

  • Validate across different plant accessions with sequence variants

  • Correlate antibody signals with mRNA expression data

  • Use CRISPR/Cas9-edited lines with epitope modifications as controls

• Combined antibody approaches:

  • Utilize a panel of antibodies targeting different epitopes

  • Implement antibody cocktails for enhanced specificity

  • Consider sandwich-based detection systems requiring recognition of multiple epitopes

The challenge of antibody cross-reactivity has been well-documented with other protein families. For instance, commercial antibodies to angiotensin receptors have shown lack of specificity leading to protein misidentification . Similar issues could arise with AT1G63350 antibodies, especially considering that the Arabidopsis genome contains numerous R-genes with high sequence similarity. The AT1G63350 protein shares significant homology with at least 16,763 proteins across 698 species, as indicated by BLAST analysis , underscoring the importance of rigorous specificity controls.

What techniques are most effective for detecting low-abundance AT1G63350 protein?

Detecting low-abundance proteins like AT1G63350, which may have variable expression levels depending on developmental stage and environmental conditions, requires specialized techniques:

• Signal amplification methods:

  • Tyramide signal amplification (TSA) for immunohistochemistry and immunofluorescence

  • Polymer-based detection systems for enhanced sensitivity in immunoblotting

  • Chemiluminescent substrates with extended signal duration

  • Proximity ligation assays for detecting protein interactions with enhanced sensitivity

• Sample enrichment strategies:

  • Immunoprecipitation prior to immunoblotting

  • Subcellular fractionation to concentrate compartment-specific signals

  • Density gradient centrifugation to isolate membrane-associated proteins

  • Size exclusion chromatography to separate protein complexes

• Advanced microscopy techniques:

  • Super-resolution microscopy for precise localization

  • Multiphoton microscopy for deeper tissue penetration

  • Fluorescence lifetime imaging to distinguish specific signals from autofluorescence

  • Light sheet microscopy for 3D visualization with minimal photobleaching

• Protocol optimizations:

  • Extended antibody incubation times at lower temperatures

  • Use of high-sensitivity detection reagents

  • Optimized blocking reagents to improve signal-to-noise ratio

  • Sample preparation methods that preserve protein integrity

The effectiveness of these techniques may vary depending on the specific properties of the AT1G63350 protein and its expression pattern. Recent advances in antibody technology, including the development of high-affinity single-domain antibodies with enhanced avidity through multimerization , offer promising approaches for detecting low-abundance proteins like AT1G63350. These engineered antibody formats can provide significant improvements in sensitivity compared to conventional antibodies.

How do post-translational modifications affect AT1G63350 antibody recognition?

Post-translational modifications (PTMs) of AT1G63350 can significantly impact antibody recognition, and understanding these effects is crucial for accurate protein detection and characterization:

• Common PTMs affecting R-proteins:

  • Phosphorylation: Often regulates R-protein activation and signaling

  • Ubiquitination: Controls protein turnover and localization

  • Myristoylation: AT1G63350 is involved in N-terminal protein myristoylation

  • SUMOylation: May regulate protein-protein interactions

• Strategies for studying PTM-dependent recognition:

  • Develop modification-specific antibodies targeting known PTM sites

  • Use λ-phosphatase treatment to assess phosphorylation-dependent recognition

  • Compare antibody binding under native vs. denaturing conditions

  • Implement 2D-gel electrophoresis to separate PTM variants

• Experimental considerations:

  • Include appropriate controls for each PTM of interest

  • Consider time-course experiments to capture dynamic modifications

  • Use PTM-preserving extraction buffers with specific inhibitors

  • Employ mass spectrometry to map actual modification sites

• Examples from other systems:

  • Phosphorylation-specific antibodies have been successfully developed for proteins involved in autophagy, such as phospho-ATG16L1, enabling monitoring of autophagy rates in cells

  • Similar approaches could be adapted for studying the phosphorylation state of AT1G63350

The development of PTM-specific antibodies requires extensive validation, but can provide valuable insights into the regulation of AT1G63350 function. For instance, antibodies that specifically recognize phosphorylated forms of ATG16L1 have been used to monitor autophagy induction in response to various stressors . Similar approaches could be employed to study how PTMs regulate AT1G63350 activity in response to pathogen challenge or environmental stresses.

What are the best protocols for using AT1G63350 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (co-IP) experiments with AT1G63350 antibodies can reveal important protein-protein interactions involved in plant immunity pathways. The following protocol recommendations are based on successful approaches with similar proteins:

• Sample preparation optimization:

  • Use mild lysis buffers to preserve protein-protein interactions (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, protease inhibitors)

  • Optimize crosslinking conditions if needed (1% formaldehyde for 10 minutes at room temperature)

  • Consider native extraction conditions for membrane-associated protein complexes

  • Include appropriate negative controls (non-immune IgG, AT1G63350 knockout tissue)

• Antibody coupling and immunoprecipitation:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Couple antibodies to beads (Protein A/G Sepharose) at optimal ratios (typically 2-5 μg antibody per 20 μl bead slurry)

  • Incubate lysates with antibody-coupled beads overnight at 4°C with gentle rotation

  • Implement stringent washing steps with increasing salt concentrations

• Analysis of co-immunoprecipitated proteins:

  • Use both silver staining and Western blotting for initial screening

  • Consider mass spectrometry for unbiased identification of interaction partners

  • Validate key interactions through reciprocal co-IP experiments

  • Confirm biological relevance through functional assays

• Specialized approaches for membrane-associated proteins:

  • Include membrane solubilization steps with appropriate detergents

  • Consider crosslinking approaches to stabilize transient interactions

  • Use density gradient centrifugation to isolate specific membrane fractions

  • Optimize buffer compositions to maintain protein complex integrity

When conducting co-IP experiments with AT1G63350 antibodies, it's important to consider that R-proteins often form dynamic complexes with other immune signaling components. These interactions may be transient or condition-dependent, necessitating careful experimental design and timing. The effectiveness of co-IP protocols will depend on the specific properties of the antibody used, including its affinity, specificity, and the accessibility of its epitope in native protein complexes.

How should immunohistochemistry protocols be optimized for AT1G63350 antibodies?

Optimizing immunohistochemistry (IHC) protocols for AT1G63350 antibodies requires careful attention to several key parameters:

• Tissue fixation and processing:

  • Compare aldehyde-based fixatives (4% paraformaldehyde, 2% glutaraldehyde) to determine optimal epitope preservation

  • Optimize fixation duration (2-24 hours) and temperature (4°C vs. room temperature)

  • Consider cryo-preservation methods to maintain native protein conformation

  • Test different embedding media (paraffin, OCT compound, LR White resin) for optimal section quality

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0)

  • Enzymatic retrieval using proteinase K or trypsin for certain epitopes

  • Determine optimal retrieval time and temperature through systematic testing

  • Consider microwave, pressure cooker, or water bath methods for consistent results

• Antibody incubation parameters:

  • Test different antibody dilutions (1:100 to 1:2000) to optimize signal-to-noise ratio

  • Compare incubation times (1 hour at room temperature vs. overnight at 4°C)

  • Evaluate different blocking reagents (normal serum, BSA, casein, commercial blockers)

  • Optimize washing procedures (buffer composition, duration, number of washes)

• Detection systems:

  • Compare direct fluorescence, polymer-based systems, and avidin-biotin complexes

  • Evaluate signal amplification methods like tyramide signal amplification

  • Test different chromogens or fluorophores for optimal visualization

  • Implement controls to distinguish specific staining from autofluorescence

Successful IHC protocols, like those developed for pectic homogalacturonan antibodies in plant tissues , demonstrate the importance of validating antibody performance under different treatment conditions. Similar validation approaches should be applied when optimizing IHC protocols for AT1G63350 antibodies. This includes testing the antibody's sensitivity to enzyme and chemical pre-treatments of the tissue sections, which can help determine the accessibility of the epitope under different conditions.

What approaches are recommended for troubleshooting weak or inconsistent AT1G63350 antibody signals?

When faced with weak or inconsistent signals when using AT1G63350 antibodies, researchers should implement a systematic troubleshooting approach:

• Sample preparation assessment:

  • Verify protein extraction efficiency through total protein quantification

  • Test alternative extraction buffers with different detergent compositions

  • Evaluate protein degradation by time-course experiments

  • Check for interfering compounds in plant extracts (phenolics, polysaccharides)

• Protocol optimization strategies:

  • Increase protein loading for Western blots (50-100 μg)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Reduce washing stringency while maintaining specificity

  • Test different membrane types (PVDF vs. nitrocellulose) for optimal protein binding

• Signal enhancement methods:

  • Implement signal amplification systems (biotin-streptavidin, tyramide)

  • Use high-sensitivity chemiluminescent substrates

  • Consider antibody concentration through affinity purification

  • Explore alternative detection systems (fluorescence vs. chromogenic)

• Control experiments to identify specific issues:

  • Include recombinant AT1G63350 protein as a positive control

  • Test the antibody against plant samples treated to induce AT1G63350 expression

  • Perform epitope competition assays to confirm specific binding

  • Compare results across different antibody lots if available

The approach to troubleshooting should be informed by an understanding of AT1G63350's biological properties. For instance, R-gene expression levels can vary significantly depending on environmental conditions , which might explain inconsistent detection in different samples. Additionally, considering that avidity engineering has been successfully employed to enhance antibody performance in other systems , similar approaches might be beneficial for improving AT1G63350 antibody signals if conventional troubleshooting methods prove insufficient.

How can researchers use AT1G63350 antibodies to study protein-protein interactions in defense signaling?

AT1G63350 antibodies can be powerful tools for elucidating protein-protein interactions in plant defense signaling networks through several methodological approaches:

• Co-immunoprecipitation-based methods:

  • Standard co-IP followed by immunoblotting for suspected interaction partners

  • Tandem affinity purification (TAP) for complex protein assemblies

  • Proximity-dependent biotin identification (BioID) to capture transient interactions

  • QUICK (quantitative immunoprecipitation combined with knockdown) to assess interaction specificity

• Microscopy-based interaction studies:

  • Immunofluorescence co-localization analysis

  • Förster resonance energy transfer (FRET) between labeled antibodies

  • Proximity ligation assay (PLA) for in situ interaction detection

  • Single-molecule localization microscopy for nanoscale interaction mapping

• Protein complex analysis techniques:

  • Blue native PAGE followed by immunoblotting

  • Size exclusion chromatography combined with immunodetection

  • Density gradient centrifugation with fraction analysis

  • Cross-linking mass spectrometry (XL-MS) for structural arrangement

• Functional validation approaches:

  • Mutual dependency analysis through genetic knockdowns

  • Domain-specific antibodies to map interaction interfaces

  • Competition assays with synthetic peptides

  • Reconstitution experiments with purified components

When studying AT1G63350 interactions, it's important to consider the dynamic nature of R-protein complexes, which often undergo conformational changes upon pathogen recognition. This may require capturing interactions at different stages of the immune response or under specific elicitor treatments. Additionally, given that R-proteins like AT1G63350 can form homo- and hetero-oligomers, careful experimental design is needed to distinguish between these different interaction types.

What are the considerations for using AT1G63350 antibodies in different plant species?

Using AT1G63350 antibodies across different plant species requires careful consideration of several factors:

• Sequence conservation analysis:

  • Perform phylogenetic analysis of AT1G63350 homologs across target species

  • Identify epitope conservation through multiple sequence alignments

  • Assess potential cross-reactivity with related R-proteins in target species

  • Consider generating species-specific antibodies for divergent homologs

• Validation strategies for cross-species applications:

  • Test antibody reactivity against recombinant proteins from different species

  • Perform epitope mapping to confirm binding site conservation

  • Use heterologous expression systems to verify specificity

  • Include appropriate positive and negative controls from each species

• Protocol adaptations for different plant materials:

  • Optimize extraction buffers for species-specific biochemical differences

  • Adjust sample preparation to account for tissue-specific interfering compounds

  • Modify fixation protocols for species with different cell wall compositions

  • Calibrate antigen retrieval methods for different tissue types

• Alternative approaches when cross-reactivity is limited:

  • Consider epitope tagging of the homologous genes in non-model species

  • Develop custom antibodies against conserved regions

  • Use heterologous expression of the target protein as a reference

  • Implement complementary approaches (e.g., transcriptomics) to support antibody data

What expression patterns have been documented for AT1G63350 in different tissues and conditions?

According to available data, AT1G63350 shows specific expression patterns that researchers should consider when designing experiments with AT1G63350 antibodies:

• Tissue-specific expression:

  • AT1G63350 has been reported to be expressed in 6 distinct plant structures

  • Expression has been detected during specific developmental stages, including:

    • LP.06 (six leaves visible)

    • LP.04 (four leaves visible)

    • Stage 4 (anthesis)

    • LP.08 (eight leaves visible)

• Regulation under stress conditions:

  • R-gene expression, including genes similar to AT1G63350, can be modulated by pathogen challenge

  • Expression levels may vary in response to environmental factors, as suggested by studies linking R-gene expression to climate variables

  • These include minimum temperature in the coldest month, number of consecutive cold days, relative humidity in spring, temperature seasonality, and precipitation in the driest month

Understanding these expression patterns is crucial for experimental design when using AT1G63350 antibodies. Researchers should consider timing their experiments to coincide with developmental stages or conditions where AT1G63350 expression is expected to be highest. Additionally, positive controls from tissues with known expression and negative controls from tissues or conditions with minimal expression should be included in experimental designs.

What reference data is available for validating AT1G63350 antibodies?

While establishing validated antibodies for AT1G63350, researchers can utilize the following reference data:

Table 1: AT1G63350 Protein Properties for Antibody Validation

Table 2: Recommended Positive and Negative Controls for AT1G63350 Antibody Validation

This reference data provides a foundation for rigorous validation of AT1G63350 antibodies. When designing validation experiments, researchers should consider both the physical properties of the protein and its biological context. Comparing antibody performance against these benchmarks will help establish confidence in antibody specificity and sensitivity.

How does AT1G63350 compare to other disease resistance proteins for antibody development?

When developing antibodies against AT1G63350, it's valuable to understand how this protein compares to other disease resistance proteins that have been successfully targeted with antibodies:

Table 3: Comparison of AT1G63350 with Other Disease Resistance Proteins Relevant to Antibody Development

R-genes like AT1G63350 present unique challenges for antibody development compared to other protein classes. Studies have shown that R-gene expression is tightly regulated and can vary significantly depending on environmental conditions . This variable expression pattern means that antibody validation should be performed under conditions where the protein is known to be expressed, possibly including treatments that induce expression.

The sequence similarity between AT1G63350 and other R-proteins necessitates careful epitope selection. Approaches similar to those used for generating antibodies against conserved sequences in other protein families, such as the AT1 receptor , may be applicable. These strategies include targeting unique regions while avoiding highly conserved functional domains that could lead to cross-reactivity.