At1g50180 Antibody

What is At1g50180 and why are antibodies against it valuable for plant immunity research?

At1g50180 is classified as a putative disease resistance protein first identified in Arabidopsis thaliana (hence the "At" prefix), with homologs present in other plant species including Nicotiana tabacum (common tobacco). In tobacco, the gene LOC107802377 encodes a putative disease resistance protein that shows homology to At1g50180 .

Antibodies against At1g50180 are valuable research tools because:

• They enable study of disease resistance protein localization during immune responses

• They facilitate investigation of protein-protein interactions in plant immunity pathways

• They allow detection of post-translational modifications that may regulate protein activity

• They support comparative studies across different plant species containing At1g50180 homologs

• They enable quantitative analysis of protein expression levels during pathogen challenges

As a putative disease resistance protein, At1g50180 likely participates in effector-triggered immunity (ETI) pathways, making antibodies against it essential for understanding molecular mechanisms of plant disease resistance.

What are the recommended approaches for validating At1g50180 antibody specificity?

Thorough validation is critical when using antibodies against plant proteins like At1g50180, which may share structural similarities with other plant disease resistance proteins. A multi-faceted validation approach should include:

Table 1: Essential Validation Steps for At1g50180 Antibodies

When validating antibodies against the tobacco homolog, researchers should note that the protein encoded by LOC107802377 (XP_016481349.1) is the putative disease resistance protein that corresponds to At1g50180 . Comprehensive validation documentation should accompany any published research using these antibodies.

What expression systems are most effective for generating At1g50180 antigens for antibody production?

The choice of expression system significantly impacts the quality of At1g50180 antigens and resulting antibodies. Several systems offer different advantages:

• E. coli expression system:

  • Advantages: High yield, cost-effective, established protocols

  • Limitations: Lacks eukaryotic post-translational modifications, potential folding issues

  • Best for: Linear epitopes, protein fragments

• Insect cell expression:

  • Advantages: Eukaryotic folding machinery, some post-translational modifications

  • Limitations: More costly than bacterial systems, moderate yield

  • Best for: Full-length protein, conformational epitopes

• Plant-based expression:

  • Advantages: Native folding environment, appropriate post-translational modifications

  • Limitations: Lower yield, more complex purification

  • Best for: Conformational antibodies, recognizing native protein

• Cell-free systems:

  • Advantages: Rapid production, control over reaction conditions

  • Limitations: Higher cost, scaling challenges

  • Best for: Difficult-to-express protein domains, toxic proteins

When using recombinant approaches for At1g50180 from tobacco, the full ORF sequence (2652bp) can be obtained from expression vectors containing the complete coding sequence . For optimal results, researchers should consider the downstream application requirements when selecting an expression system.

How should researchers optimize immunohistochemistry protocols for At1g50180 detection in plant tissues?

Immunohistochemical detection of At1g50180 in plant tissues requires protocol optimization to address unique challenges of plant material:

Sample preparation:

• Fix tissues in 4% paraformaldehyde for 12-24 hours at 4°C

• Consider vacuum infiltration to improve fixative penetration

• Optimize embedding medium (paraffin vs. cryosectioning) based on epitope sensitivity

• Section thickness typically 5-10 μm for optimal antibody penetration

Antigen retrieval:

• Heat-induced epitope retrieval in citrate buffer (pH 6.0) often improves detection

• Enzymatic retrieval with proteinase K may be necessary for some fixed tissues

• Test multiple retrieval methods to determine optimal conditions

Blocking and antibody incubation:

• Use 2-5% BSA with 0.3% Triton X-100 in PBS for blocking (2 hours at room temperature)

• Dilute primary antibody in blocking solution (1:100-1:500 range)

• Incubate sections with primary antibody overnight at 4°C

• Wash extensively (5-6 times, 10 minutes each) with PBS containing 0.1% Tween-20

Signal detection:

• Fluorescent secondary antibodies generally provide better signal-to-noise ratio than enzymatic detection

• Choose fluorophores that avoid overlap with plant autofluorescence (far-red dyes often work well)

• Include DAPI or other nuclear counterstains for tissue orientation

• Acquire images using confocal microscopy for optimal resolution

Each step should be systematically optimized, testing multiple conditions in parallel to determine the protocol that yields specific signal with minimal background.

What strategies help overcome cross-reactivity challenges with At1g50180 antibodies?

Cross-reactivity with related plant disease resistance proteins represents a significant challenge for At1g50180 antibodies. These approaches can help address this issue:

• Epitope selection strategies:

  • Target unique regions rather than conserved NBS-LRR domains

  • Perform sequence alignments to identify At1g50180-specific regions

  • Consider the variable C-terminal regions for antibody generation

• Antibody purification approaches:

  • Perform affinity purification against the specific immunizing antigen

  • Consider negative selection against closely related proteins

  • Use sequential purification steps to remove cross-reactive antibodies

• Experimental controls:

  • Include tissues lacking At1g50180 expression as negative controls

  • Perform peptide competition assays to confirm specificity

  • Validate results using complementary detection methods (e.g., mass spectrometry)

• Signal validation:

  • Compare antibody signal patterns with transcript expression data

  • Use genetic approaches (knockdown/knockout) to confirm specificity

  • Include multiple antibodies targeting different epitopes

Table 2: Domains of At1g50180 and Cross-reactivity Considerations

By combining these approaches, researchers can develop experimental protocols that maximize specific detection while minimizing cross-reactivity issues.

How does plant developmental stage affect At1g50180 antibody detection efficiency?

Plant developmental stage significantly impacts At1g50180 detection due to expression level variations, protein modifications, and tissue composition changes:

• Expression level considerations:

  • Disease resistance proteins often show developmental regulation

  • Expression may increase during reproductive stages or under stress

  • Basal expression levels in vegetative tissues may be below detection limits

• Tissue accessibility factors:

  • Younger tissues generally offer better antibody penetration

  • Older tissues may require more aggressive extraction methods

  • Secondary cell wall development can impede antibody access

  • Increased vacuolar volume in mature cells may dilute protein concentration

• Interfering compounds:

  • Secondary metabolites accumulate with development and may interfere with antibody binding

  • Phenolic compounds in mature tissues can cross-link with proteins

  • Extraction buffer composition should be adjusted based on tissue type and age

• Protocol adjustments by developmental stage:

  • Seedlings: Standard extraction buffers with mild detergents

  • Vegetative tissues: Include PVPP to remove phenolics

  • Reproductive tissues: Consider specialized buffers with higher detergent concentrations

  • Senescent tissues: Add additional protease inhibitors to prevent degradation

For developmental studies, researchers should optimize extraction and detection protocols for each specific stage, potentially establishing stage-specific baseline detection parameters.

How can At1g50180 antibodies be used to investigate protein-protein interactions in plant immunity?

At1g50180 antibodies enable multiple approaches for studying protein-protein interactions that are crucial for understanding disease resistance signaling:

• Co-immunoprecipitation (Co-IP):

  • Use At1g50180 antibodies to pull down protein complexes

  • Analyze interactions before and after pathogen challenge

  • Couple with mass spectrometry for unbiased interactome analysis

  • Validate specific interactions with reverse Co-IP

• Proximity labeling approaches:

  • Use antibodies to validate expression of At1g50180 fused to BioID or APEX2

  • Identify neighbors in the protein interaction network

  • Compare interactomes under different conditions

• Microscopy-based interaction studies:

  • Immunofluorescence co-localization with potential partners

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

  • FRET analysis of tagged proteins validated with antibodies

Table 3: Protein-Protein Interaction Methods for At1g50180 Research

When designing interaction studies, researchers should consider the potential dynamic nature of At1g50180 interactions during immune responses, potentially investigating time courses following pathogen challenge.

What approaches allow investigation of At1g50180 post-translational modifications?

Post-translational modifications (PTMs) likely play critical roles in regulating At1g50180 function during immune responses. These approaches enable their investigation:

• PTM-specific antibody development:

  • Generate antibodies against predicted phosphorylation sites

  • Develop antibodies recognizing ubiquitinated or SUMOylated forms

  • Validate using in vitro modified recombinant proteins

• IP-based approaches:

  • Immunoprecipitate At1g50180 using specific antibodies

  • Analyze by mass spectrometry to identify modifications

  • Compare PTM profiles before and after immune activation

  • Use phosphatase treatments to confirm phosphorylation

• Gel mobility shift analysis:

  • Compare migration patterns of modified and unmodified forms

  • Use Phos-tag gels to enhance separation of phosphorylated species

  • Apply deubiquitinating enzymes to confirm ubiquitination

• Site-directed mutagenesis validation:

  • Generate mutants at putative modification sites

  • Express in planta and validate with antibodies

  • Compare with wild-type protein under inducing conditions

Table 4: Common PTMs for Disease Resistance Proteins Like At1g50180

When investigating PTMs, time-course experiments are particularly valuable, as modifications may be transient during signaling events.

How can researchers use At1g50180 antibodies to study its role in plant-pathogen interactions?

At1g50180 antibodies provide powerful tools for investigating the protein's function during plant-pathogen interactions:

• Spatial dynamics during infection:

  • Track protein localization before and after pathogen challenge

  • Examine accumulation at infection sites

  • Monitor subcellular redistribution during immune responses

  • Compare compatible vs. incompatible interactions

• Temporal dynamics of protein abundance:

  • Quantify At1g50180 levels at different timepoints after infection

  • Correlate protein levels with defense gene activation

  • Monitor protein stability during sustained infections

  • Compare responses to different pathogen types

• Interaction with pathogen effectors:

  • Use antibodies to study co-localization with pathogen effectors

  • Perform Co-IP to detect direct or indirect effector interactions

  • Identify effector-induced modifications

  • Study structural changes upon effector perception

• Genetic complementation studies:

  • Transform susceptible plants with At1g50180 variants

  • Use antibodies to confirm protein expression

  • Correlate protein abundance with resistance phenotypes

  • Identify critical domains through mutation analysis

These approaches can be integrated to develop comprehensive models of At1g50180 function during plant immune responses, potentially revealing mechanisms that could be targeted for enhancing disease resistance in crops.

What are the optimal conditions for Western blot detection of At1g50180?

Western blot detection of At1g50180 requires careful optimization due to its potentially low expression levels and structural characteristics:

Sample preparation:

• Use extraction buffer containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% Triton X-100, 1mM EDTA

• Include protease inhibitor cocktail, 1mM DTT, and 1mM PMSF

• Consider adding phosphatase inhibitors if studying phosphorylation

• Homogenize tissue thoroughly at 4°C and clarify by centrifugation

Gel electrophoresis:

• Use 8% polyacrylamide gels for optimal resolution of large proteins

• Load 50-75 μg total protein per lane

• Include positive control samples when available

• Use pre-stained molecular weight markers

Transfer conditions:

• Transfer to PVDF membrane (0.45 μm pore size)

• Use wet transfer at 30V overnight at 4°C for high molecular weight proteins

• Consider adding 0.05% SDS to transfer buffer for efficient transfer

• Verify transfer efficiency with reversible staining (Ponceau S)

Antibody incubation:

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

• Dilute primary antibody 1:1000 in 5% BSA in TBST

• Incubate with primary antibody overnight at 4°C with gentle agitation

• Wash 4-5 times with TBST (10 minutes each)

• Use HRP-conjugated secondary antibody at 1:5000-1:10000 dilution

Detection:

• Use enhanced chemiluminescence for sensitive detection

• Consider using signal enhancer systems for low abundance proteins

• Optimize exposure time to avoid signal saturation

• Include appropriate loading controls (anti-actin, anti-tubulin)

Table 5: Troubleshooting Western Blot Issues for At1g50180

How can researchers overcome challenges in immunoprecipitating At1g50180?

Immunoprecipitation (IP) of At1g50180 presents challenges due to potential low abundance and complex interactions:

• Optimized lysis conditions:

  • Test multiple lysis buffers with varying detergent concentrations

  • For membrane-associated forms, consider NP-40 (0.5-1%) or digitonin (1%)

  • Include protease inhibitors and phosphatase inhibitors

  • Perform lysis at 4°C with gentle agitation

• Antibody selection and coupling:

  • Compare polyclonal vs. monoclonal antibodies for IP efficiency

  • Test direct coupling to beads vs. protein A/G capture

  • Optimize antibody amount (1-5 μg per reaction)

  • Consider crosslinking antibody to beads to prevent co-elution

• Pre-clearing strategy:

  • Pre-clear lysate with beads alone to reduce non-specific binding

  • Include controls with non-specific IgG

  • Consider two-step pre-clearing for highly complex samples

• Washing optimization:

  • Start with lower stringency washes (150mM NaCl)

  • Perform sequential washes with increasing stringency

  • Optimize wash buffer composition (salt, detergent)

  • Determine optimal number of washes (typically 4-6)

• Elution methods:

  • Compare different elution strategies:

    • Denaturing: SDS sample buffer at 95°C

    • Non-denaturing: Glycine pH 2.5, peptide competition

  • For functional studies, use gentle elution to preserve activity

  • For mass spectrometry, consider on-bead digestion

By systematically optimizing these parameters, researchers can develop robust IP protocols for studying At1g50180 and its interaction partners.

What strategies help overcome low expression challenges when detecting At1g50180?

At1g50180 and related disease resistance proteins often have low basal expression levels. These strategies can enhance detection:

• Sample enrichment approaches:

  • Perform subcellular fractionation to concentrate the protein

  • Use immunoprecipitation prior to analysis

  • Implement protein concentration methods (TCA precipitation)

  • Consider size exclusion filtration to eliminate abundant proteins

• Expression enhancement strategies:

  • Induce expression with pathogen treatment or elicitors

  • Use defense signaling molecules (salicylic acid, jasmonic acid)

  • Select tissues with higher basal expression

  • Consider developmental stages with elevated expression

• Signal amplification techniques:

  • Implement tyramide signal amplification for immunohistochemistry

  • Use enhanced chemiluminescence substrates for Western blots

  • Apply biotin-streptavidin amplification systems

  • Consider polymer-based detection systems

• Specialized extraction methods:

  • Implement sequential extraction to isolate different protein pools

  • Use specialized buffers for membrane-associated proteins

  • Add protein stabilizers to prevent degradation

  • Include compounds to remove interfering substances

Table 6: Detection Limit Comparison for Plant Resistance Proteins

By combining multiple approaches (e.g., sample enrichment followed by sensitive detection), researchers can overcome the challenges of detecting low-abundance proteins like At1g50180.

How can synthetic antibody technologies enhance At1g50180 research?

Emerging synthetic antibody technologies offer new possibilities for studying challenging targets like At1g50180:

• Recombinant antibody fragments:

  • Single-chain variable fragments (scFv) provide smaller size for tissue penetration

  • Fab fragments eliminate Fc-mediated background in plant tissues

  • Can be produced in bacterial systems for consistent quality

  • Allow for site-specific labeling for advanced imaging

• Nanobodies (VHH antibodies):

  • Derived from camelid single-domain antibodies

  • Extremely small size (~15 kDa) for accessing restricted epitopes

  • High stability in varied conditions

  • Potential for intracellular expression in planta

• Aptamer alternatives:

  • Nucleic acid-based recognition molecules

  • Can be evolved for high specificity

  • Reversible binding with controlled dissociation

  • Potential for in vivo sensors

• In planta antibody expression:

  • Express antibody fragments directly in plant tissues

  • Create fusion reporters for real-time monitoring

  • Develop sensors for protein conformation changes

  • Study protein dynamics without fixation artifacts

• Computational design approaches:

  • Structure-based antibody engineering

  • In silico epitope prediction and optimization

  • Machine learning approaches for specificity enhancement

  • Rational design of plant-optimized antibody formats

These technologies address traditional limitations of antibodies in plant research, particularly for challenging targets like membrane-associated disease resistance proteins. Their implementation requires specialized expertise but offers significant advantages for advanced applications.

How can At1g50180 antibody studies be integrated with -omics approaches?

Integration of antibody-based approaches with -omics technologies creates powerful systems for comprehensive understanding of At1g50180 function:

• Antibody-proteomics integration:

  • Use antibodies for targeted protein complex isolation

  • Combine with mass spectrometry for interactome analysis

  • Compare protein complexes across conditions or treatments

  • Identify post-translational modifications

• Spatial transcriptomics correlation:

  • Perform immunohistochemistry on tissue sections

  • Correlate protein localization with local transcriptomes

  • Map spatial relationships between At1g50180 and defense gene activation

  • Develop spatial models of immune activation

• Single-cell approaches:

  • Use antibodies for cell sorting of At1g50180-expressing cells

  • Perform single-cell transcriptomics or proteomics

  • Identify cell-type specific functions

  • Map cellular heterogeneity in immune responses

• Systems biology integration:

  • Use antibody-derived protein interaction data as network nodes

  • Integrate with transcriptomic responses to pathogens

  • Develop dynamic models of resistance protein function

  • Generate testable hypotheses for experimental validation

Table 7: Integration of At1g50180 Antibody Applications with -Omics Approaches

By thoughtfully designing experiments that leverage both antibody-based approaches and -omics technologies, researchers can develop more comprehensive understanding of At1g50180 function in plant immunity.

How will antibody research contribute to translating At1g50180 findings to crop improvement?

Antibody-based research on At1g50180 and related disease resistance proteins provides critical insights for crop improvement strategies:

• Functional validation in crops:

  • Use antibodies to validate expression of resistance genes in transgenic crops

  • Confirm protein stability and localization in elite varieties

  • Monitor protein levels in different tissues and developmental stages

  • Correlate protein abundance with disease resistance phenotypes

• Resistance mechanism characterization:

  • Study protein dynamics during pathogen infection in crop species

  • Compare immune complex formation between model and crop plants

  • Identify conserved and divergent aspects of resistance mechanisms

  • Develop models for engineering more effective resistance

• Breeding program applications:

  • Develop antibody-based screening tools for resistant germplasm

  • Create high-throughput assays for protein expression levels

  • Monitor protein variants across breeding populations

  • Validate resistance protein function in hybrids

• Engineered resistance evaluation:

  • Confirm expression of engineered resistance proteins

  • Verify protein folding and stability of modified variants

  • Study interaction with endogenous immune components

  • Monitor protein behavior under field conditions

Through these applications, antibody-based research provides a crucial bridge between basic understanding of resistance protein function and applied efforts to enhance crop disease resistance, potentially contributing to more sustainable agricultural practices and improved food security.