At1g51480 Antibody

What is AT1G51480 and why would researchers need antibodies against it?

AT1G51480 encodes a disease resistance protein belonging to the CC-NBS-LRR (coiled-coil nucleotide-binding site leucine-rich repeat) family in Arabidopsis thaliana. According to subcellular localization data, this protein is primarily found in the cytosol with a SUBAcon score of 0.945 . It has a molecular weight of approximately 107.4 kDa and an isoelectric point of 6.29 .
Researchers need antibodies against AT1G51480 for several important applications:

• Studying subcellular localization during plant immune responses

• Examining protein expression patterns during pathogen infection

• Investigating protein-protein interactions in defense signaling pathways

• Analyzing post-translational modifications that may regulate its function

• Determining its role in specific defense response pathways through functional analyses
  The CC-NBS-LRR class of proteins is particularly important as they represent a major class of plant disease resistance genes that have undergone positive selection during evolution, as demonstrated in genome-wide analyses .

What validation strategies should be used for AT1G51480 antibodies?

Proper validation of AT1G51480 antibodies is critical for ensuring experimental reliability. Multiple validation approaches should be employed:

Primary Validation Strategies:

• Genetic Validation: Testing on tissues from AT1G51480 knockout mutants is the gold standard. This approach is critical as search result demonstrates that some commercial antibodies lack specificity and may recognize proteins other than their intended targets.

• Orthogonal Validation: Compare antibody detection with transcript expression data (RT-PCR or RNA-seq). This approach verifies that protein detection correlates with known mRNA expression patterns .

• Independent Antibody Validation: Use two antibodies targeting different regions of AT1G51480. This strategy is particularly effective when antibodies targeting non-overlapping epitopes show similar detection patterns .

• Expression Validation: Test on tissues with differential expression or in plants where AT1G51480 is overexpressed.

What experimental approaches are most effective for immunolocalization of AT1G51480 in plant tissues?

For effective immunolocalization of AT1G51480 in Arabidopsis tissues, researchers should consider:

Tissue Preparation:

• Utilize the whole-mount immunolocalization protocol described in search result , which preserves tissue architecture while allowing antibody penetration

• Consider cell wall digestion or permeabilization steps to improve antibody access

• Test multiple fixation methods to ensure epitope preservation (paraformaldehyde vs. glutaraldehyde)

Detection Methods:

• For confocal microscopy: Use fluorophore-conjugated secondary antibodies with appropriate controls

• For electron microscopy: Consider immunogold labeling as demonstrated in search result , which successfully localized cytosolic proteins ACBP4 and ACBP5 in Arabidopsis

• Include counterstains like propidium iodide to visualize cell structures

Critical Controls:

• No primary antibody control

• Pre-immune serum control

• Peptide competition assay (pre-absorbing antibody with immunizing peptide)

• Tissue from AT1G51480 knockout plants

• Autofluorescence controls (particularly important in plant tissues)
  The protocol in search result specifically notes that their approach "preserves the constitution of the developing primordium and incorporates the architecture of the ovule," which is valuable for maintaining cellular context during immunolocalization studies.

How should Western blot protocols be optimized for AT1G51480 detection?

Optimizing Western blot protocols for AT1G51480 detection requires addressing several key considerations:

Sample Preparation:

• Use extraction buffers containing protease inhibitors to prevent degradation

• Include reducing agents to ensure proper protein denaturation

• Consider multiple detergent types for optimal solubilization

• Test different tissue types and developmental stages where AT1G51480 is expressed

Technical Parameters:

Critical Controls:

• Include recombinant AT1G51480 protein or overexpression line as positive control

• Use tissue from AT1G51480 knockout mutant as negative control

• Include loading controls appropriate for plant tissues
  Search result provides guidance on presenting Western blot data in a scientific manner, emphasizing the importance of showing representative images with appropriate molecular weight markers and controls.

How can researchers distinguish between AT1G51480 and other CC-NBS-LRR proteins using antibodies?

Distinguishing AT1G51480 from other related CC-NBS-LRR proteins presents a significant challenge due to sequence similarities. Effective strategies include:

Epitope Selection:

• Target unique regions with low homology to other family members

• Avoid conserved domains like the NBS region

• Focus on the variable LRR region, which often undergoes positive selection

• According to search result , AT1G51480's closest protein match is AT5G43730.1, so epitopes should be selected to avoid cross-reactivity

Antibody Development:

• Consider monoclonal antibodies for highest specificity

• Use affinity purification against the specific epitope

• Test against a panel of related proteins to confirm specificity

• Perform pre-absorption with related proteins to remove cross-reactive antibodies

Validation Approaches:

• Test on knockout mutants of AT1G51480 and related genes

• Compare detection patterns with known expression profiles of different family members

• Use peptide competition assays with specific and related epitopes

• Perform immunoprecipitation followed by mass spectrometry to confirm target identity
  Search result describes enhanced validation approaches that can be adapted to ensure antibody specificity when dealing with protein families with high sequence similarity.

What factors influence the subcellular localization detection of AT1G51480?

The accurate detection of AT1G51480's subcellular localization depends on several factors:

Technical Considerations:

• Fixation methods significantly impact epitope preservation and accessibility

• Permeabilization conditions affect antibody penetration into cellular compartments

• Detection sensitivity may be crucial for visualizing low-abundance proteins

• Cell wall composition varies between tissues and can affect antibody penetration

Biological Factors:

• AT1G51480 is predicted to be cytosolic (SUBAcon score 0.945) , but may relocalize during immune responses

• Protein-protein interactions may mask epitopes in certain cellular contexts

• Post-translational modifications could affect localization patterns

• Expression levels may vary based on developmental stage or stress conditions

Verification Methods:

• Compare antibody detection with fluorescently-tagged AT1G51480 localization

• Use different fixation and permeabilization protocols to confirm consistent localization

• Employ subcellular fractionation followed by Western blot as a complementary approach

• Consider electron microscopy for high-resolution localization studies
  Search result demonstrates how researchers successfully employed both biochemical fractionation and immunoelectron microscopy to confirm the cytosolic localization of related Arabidopsis proteins ACBP4 and ACBP5.

How do protein extraction methods affect AT1G51480 antibody detection?

Protein extraction methods significantly impact the detection of AT1G51480 by antibodies:

Buffer Composition Effects:

Extraction Challenges:

• Plant tissues contain compounds that can interfere with antibody binding

• Cell wall components may reduce extraction efficiency

• High levels of proteases in plant tissues require effective inhibition

• Secondary metabolites may cause protein modifications that affect epitope recognition

Optimization Strategies:

• Test multiple extraction protocols and compare recovery

• Consider phenol extraction for highly recalcitrant tissues

• Use subcellular fractionation to enrich for cytosolic proteins

• Implement quality control measures to assess protein integrity after extraction
  According to search result , successful detection of cytosolic proteins in Arabidopsis required careful optimization of extraction conditions and verification through multiple independent methods.

What role do post-translational modifications play in AT1G51480 antibody recognition?

Post-translational modifications (PTMs) can significantly affect antibody recognition of AT1G51480:

Common PTMs in Plant Resistance Proteins:

• Phosphorylation: Critical for activation of defense signaling

• Ubiquitination: Regulates protein turnover and function

• SUMOylation: Affects protein localization and activity

• Glycosylation: Less common in cytosolic proteins but may occur

Impact on Antibody Binding:

• PTMs may directly block antibody access to epitopes

• Conformational changes induced by PTMs can expose or hide epitopes

• Different modification states may represent functionally distinct protein pools

• Modification-specific antibodies can help study activation states

Experimental Considerations:

• Include phosphatase inhibitors when studying phosphorylated forms

• Compare antibody binding before and after phosphatase treatment

• Consider developing modification-specific antibodies for functional studies

• Document extraction conditions that may affect PTM status
  Search result indicates that angiotensin receptor type 1 (AT1R) antibodies could detect different activation states of receptors, suggesting similar approaches might be valuable for studying AT1G51480 activation during immune responses.

How can researchers develop a quantitative ELISA for AT1G51480 protein?

Developing a quantitative ELISA for AT1G51480 requires systematic optimization:

ELISA Format Selection:

• Sandwich ELISA: Requires two antibodies recognizing different epitopes

• Competitive ELISA: Useful when only one antibody is available

• Direct ELISA: Simplest approach but may have higher background

Development Steps:

• Antibody Characterization:

  • Test antibody specificity and sensitivity

  • Determine optimal coating concentration

  • Validate with positive and negative controls

• Standard Curve Development:

  • Produce and purify recombinant AT1G51480 protein

  • Create dilution series for calibration

  • Verify linearity across relevant concentration range

• Sample Preparation Optimization:

  • Test different extraction buffers

  • Determine matrix effects from plant extracts

  • Develop consistent homogenization protocol

• Protocol Validation:

  • Determine limit of detection and quantification

  • Assess inter- and intra-assay variability

  • Perform spike recovery tests to confirm accuracy

Performance Metrics:

• Sensitivity: Lower limit of detection should be below physiological levels

• Specificity: No cross-reactivity with related CC-NBS-LRR proteins

• Reproducibility: CV < 15% between technical replicates

• Accuracy: Recovery of 80-120% in spike-recovery experiments
  Search result describes quantitative ELISA development for measuring autoantibodies, providing methodological principles that can be adapted for plant protein quantification.

What approaches enable investigation of AT1G51480 protein-protein interactions?

Investigating AT1G51480 protein-protein interactions requires specialized techniques:

Co-Immunoprecipitation (Co-IP):

• Use AT1G51480 antibodies to pull down protein complexes

• Optimize buffer conditions to preserve interactions

• Include appropriate controls (IgG, knockout tissue)

• Identify interacting partners by mass spectrometry

Proximity Labeling:

• Fuse AT1G51480 to BioID or TurboID for in vivo proximity labeling

• Express in Arabidopsis to capture physiologically relevant interactions

• Purify biotinylated proteins and identify by mass spectrometry

• Validate key interactions through independent methods

Yeast Two-Hybrid (Y2H):

• Screen for interactors using AT1G51480 domains as bait

• Verify interactions through directed Y2H with candidate proteins

• Test with domain deletions to map interaction interfaces

Bimolecular Fluorescence Complementation (BiFC):

• Express AT1G51480 and candidate interactors as fusion proteins

• Visualize interactions through fluorescence complementation

• Confirm subcellular localization of interaction complexes

Considerations for AT1G51480:

• As a defense protein, interactions may be transient or condition-specific

• Consider pathogen challenge to induce relevant interactions

• Test domain-specific interactions to understand functional significance

• Verify in planta through multiple independent approaches
  Search result mentions screening for protein interactors using yeast two-hybrid systems, noting that "identification of such interactors would be expected to further enhance our knowledge" of protein function.

How can researchers study AT1G51480 expression during plant immune responses?

Studying AT1G51480 expression during immune responses requires integrated approaches:

Transcript Analysis:

• qRT-PCR for sensitive quantification of mRNA levels

• RNA-seq for genome-wide expression context

• In situ hybridization for tissue-specific localization

Protein Analysis:

• Western blotting for semi-quantitative protein detection

• Immunohistochemistry for spatial expression patterns

• ELISA for quantitative measurement across conditions

Experimental Design Considerations:

Pathogen Systems:

• Test multiple pathogen types (bacteria, fungi, oomycetes)

• Include both compatible and incompatible interactions

• Consider molecular patterns (PAMPs) and effectors separately

• Compare local and systemic responses
  Search result indicates that NBS-LRR genes like AT1G51480 are subject to positive selection, suggesting their importance in evolving plant immunity, which makes studying their expression patterns during pathogen challenge particularly relevant.

What are best practices for immunoprecipitation of AT1G51480?

Optimizing immunoprecipitation (IP) of AT1G51480 requires careful consideration of multiple factors:

Buffer Optimization:

• Test different lysis buffers (RIPA, NP-40, Digitonin)

• Optimize salt concentration (150-500 mM NaCl)

• Include appropriate protease and phosphatase inhibitors

• Consider detergent types and concentrations

Antibody Selection:

• Use affinity-purified antibodies for highest specificity

• Determine optimal antibody-to-lysate ratio

• Consider direct antibody conjugation to beads

• Validate antibody specificity through knockout controls

Protocol Refinement:

Critical Controls:

• Input sample (pre-IP lysate)

• IgG control from same species

• AT1G51480 knockout tissue

• Competing peptide elution

Downstream Analysis:

• Western blot to confirm target enrichment

• Mass spectrometry for interactome analysis

• Activity assays if functional studies are planned
  Search result demonstrates successful immunoprecipitation of Arabidopsis proteins, emphasizing the importance of buffer optimization and appropriate controls for IP validation.

How do different plant tissue types affect AT1G51480 antibody performance?

The performance of AT1G51480 antibodies can vary significantly across different plant tissue types:

Tissue-Specific Challenges:

Impact on Experimental Approaches:

• Immunohistochemistry: Cell wall composition affects fixation and permeabilization requirements

• Western blotting: Different tissues require optimized extraction and loading controls

• IP: Background proteome affects non-specific binding profiles

• ELISA: Matrix effects vary with tissue type

Optimization Strategies:

• Develop tissue-specific extraction protocols

• Validate antibody performance in each tissue type separately

• Include appropriate tissue-specific controls

• Consider subcellular fractionation to enrich for target compartment
  According to search result , tissue-specific differences in cell wall composition can significantly affect antibody penetration and binding in immunolocalization studies.

What strategies help distinguish between specific and non-specific antibody binding?

Distinguishing specific from non-specific binding is critical for reliable AT1G51480 detection:

Experimental Controls:

• Genetic Controls:

  • AT1G51480 knockout mutant tissue (gold standard negative control)

  • AT1G51480 overexpression line (positive control)

  • Related CC-NBS-LRR mutants to assess cross-reactivity

• Technical Controls:

  • Pre-immune serum or isotype control

  • No primary antibody control

  • Peptide competition assay (pre-absorption with immunizing peptide)

  • Secondary antibody only control

Binding Characteristics Analysis:

• Titration experiments to establish dose-dependent binding

• Competition with increasing concentrations of specific peptide

• Testing multiple antibodies against different epitopes

• Cross-adsorption against related proteins

Advanced Validation:

• Correlation with orthogonal detection methods (e.g., GFP tagging)

• Mass spectrometry identification of immunoprecipitated proteins

• Comparing binding patterns with known expression data
  Search result emphasizes the importance of knockout controls for antibody validation, noting that commercial antibodies can lack specificity and may recognize proteins other than their intended targets.

How can researchers use antibodies to study AT1G51480 in different Arabidopsis ecotypes?

Studying AT1G51480 across different Arabidopsis ecotypes presents both challenges and opportunities:

Sequence Variation Considerations:

• Check for polymorphisms in AT1G51480 sequence across ecotypes

• Assess whether variations affect antibody epitopes

• Consider developing ecotype-specific antibodies if necessary

• Focus on conserved regions for universal detection

Experimental Design:

Validation Approaches:

• Sequence the AT1G51480 gene in ecotypes of interest

• Test antibody binding to recombinant proteins from different ecotypes

• Include ecotype-specific controls in all experiments

• Consider complementary genomic approaches (RNA-seq, whole genome sequencing)
  As indicated in search result , Arabidopsis serves as "a model system not only for studying numerous aspects of plant biology, but also for understanding mechanisms of the rapid evolutionary process," making cross-ecotype studies particularly valuable. These FAQs provide comprehensive guidance for researchers working with AT1G51480 antibodies, addressing both basic technical considerations and advanced research applications in Arabidopsis immunity studies.