At5g45510 Antibody

What is the At5g45510 gene and what protein does it encode?

At5g45510 is a gene located on chromosome 5 of Arabidopsis thaliana that encodes NRG1C, a C-terminal truncated NLR (nucleotide-binding domain and leucine-rich repeat) protein lacking the full N-terminal coiled-coil domain. This gene belongs to the leucine-rich repeat (LRR) family protein classification according to TAIR (The Arabidopsis Information Resource) . The protein functions primarily as a negative regulator of TNL (TIR-NB-LRR)-mediated immunity by antagonizing full-length NLRs like NRG1A and NRG1B. Understanding the structure and function of this protein is essential for designing appropriate immunogens for antibody production and for interpreting experimental results when using At5g45510-specific antibodies.

How should researchers validate the specificity of At5g45510 antibodies?

Validation of At5g45510 antibodies requires a multi-step approach similar to established antibody validation protocols. Begin with structural validation through SDS-PAGE analysis to confirm the expected molecular weight bands (~25 kDa light chain and ~50 kDa heavy chain with >91% purity) . For functional validation, implement antigen-specific ELISA using purified recombinant NRG1C protein to establish binding curves and sensitivity parameters . Mass spectrometric verification is crucial to confirm monoclonal origin by analyzing distinct light chain (approximately 23742 m/z) and heavy chain (approximately 49858 m/z) signals .

To confirm cellular target specificity, perform dual-fluorochrome labeling using NRG1C protein tagged with two different fluorophores (such as AF647 and PE) for flow cytometry analysis - expect ≥99% double-positive staining in target cells when using a validated antibody . Always include genetic validation using knockout lines (nrg1c) as negative controls to confirm absence of signal in these samples.

What are the critical quality control parameters for At5g45510 antibody production?

Implementing a rigorous quality control framework for At5g45510 antibodies involves these critical parameters:

• Hybridoma validation: Verify CD138 and IgG positivity of the hybridoma cell line with ≥99% Dsg3-specific reactivity using dual-fluorophore labeling techniques .

• Purity assessment: Quantify antibody purity via SDS-PAGE, with acceptable standard purity >91% (analyzing 25 kDa light and 50 kDa heavy antibody chains versus unspecific bands) .

• Functional verification: Establish standard ELISA curves with consistent batch-to-batch performance, using purified recombinant NRG1C protein .

• Structural confirmation: Implement intact protein mass spectrometry after TCEP reduction to verify monoclonal origin through defined signals for light chain (~23742 m/z) and heavy chain (~49858 m/z) .

• Batch-to-batch variation analysis: Compare different production batches using standard functional assays to ensure consistent performance across production runs .

The implementation of these quality control steps ensures reliable antibody performance across experiments, enabling meaningful comparisons between different studies utilizing the At5g45510 antibody.

How should researchers design experiments to investigate NRG1C-mediated immune regulation?

When designing experiments to investigate NRG1C-mediated immune regulation, researchers should implement a comprehensive approach combining genetic manipulation and antibody-based detection:

Step 1: Generate experimental systems with varying NRG1C expression levels:

• Overexpression lines using strong constitutive promoters

• Knockout lines using CRISPR-Cas9 or T-DNA insertion

• Native expression controls

Step 2: Challenge these plant lines with pathogens known to be affected by NRG1C function:

• Hyaloperonospora arabidopsidis (oomycete pathogen)

• Pseudomonas syringae (bacterial pathogen)

Step 3: Implement dual-detection methods:

• Antibody-based detection of NRG1C protein levels using validated At5g45510 antibodies

• RT-qPCR for transcript level analysis

• Phenotypic assessment of plant immune responses

Step 4: Analyze interaction with known partners by co-immunoprecipitation using At5g45510 antibodies to pull down:

• SAG101

• EDS1

• NRG1A/NRG1B

For accurate quantification of protein-protein interactions, TurboID-based proximity labeling with HA-tagged NRG1C fusion proteins can be detected using anti-HA antibodies.

The table below summarizes expected phenotypes in different genetic backgrounds:

What are the optimal conditions for At5g45510 antibody use in immunoblotting applications?

For optimal performance of At5g45510 antibodies in immunoblotting applications, researchers should follow this methodological approach:

Sample preparation:

• Extract total protein from plant tissue using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, and protease inhibitor cocktail

• Quantify protein concentration using Bradford or BCA assay

• Denature samples with reducing agent (5 mM TCEP) for 60 minutes at room temperature

Gel electrophoresis parameters:

• Load 10-20 μg protein per lane on 10-12% SDS-PAGE gels

• Include positive controls (recombinant NRG1C) and negative controls (protein extract from nrg1c knockout)

• Run at 100V for stacking gel and 150V for resolving gel

Transfer and antibody incubation:

• Transfer proteins to PVDF membrane (0.45 μm) at 100V for 60 minutes

• Block membrane with 5% non-fat dry milk or BSA in TBST for 1 hour

• Incubate with primary At5g45510 antibody at 2.5 μg/ml concentration (optimal dilution should be determined empirically for each batch)

• Wash 3x with TBST

• Incubate with species-specific HRP-conjugated secondary antibody (1:2000 dilution)

• Develop using ECL substrate and capture images with digital imaging system

Expected results:

• Primary band at approximately the predicted molecular weight of NRG1C

• Absence of this band in negative control samples

• Confirmation of specificity through peptide competition assays

How can At5g45510 antibodies be employed to investigate NRG1C's role in autoimmunity suppression?

To investigate NRG1C's role in autoimmunity suppression using At5g45510 antibodies, implement this methodological framework:

Genetic system preparation:

• Generate stable transgenic lines with varying NRG1C expression in autoimmune backgrounds:

  • chs3-2D mutants (where NRG1C overexpression fully suppresses autoimmune phenotypes)

  • snc1 mutants (where NRG1C overexpression partially suppresses dwarfism)

  • Wild-type controls

Protein expression analysis:

• Perform immunoblotting with At5g45510 antibodies to quantify NRG1C protein levels across genotypes

• Implement immunohistochemistry to determine tissue-specific expression patterns

• Use dual-fluorochrome labeling with flow cytometry to quantify cellular distribution

Interaction studies:

• Conduct co-immunoprecipitation experiments using At5g45510 antibodies to identify protein complexes

• Perform proximity labeling using TurboID-fused NRG1C to identify transient interactors

• Execute competitive binding assays to test NRG1C antagonism against NRG1A/B

Functional assessment:

• Measure morphological parameters (plant height, leaf size) to quantify dwarfism suppression

• Challenge plants with pathogens to assess disease resistance

• Analyze gene expression profiles of known defense response genes

• Quantify cellular immune markers using validated antibody panels in flow cytometry

This comprehensive approach allows researchers to correlate NRG1C protein levels with autoimmunity suppression phenotypes, while identifying key molecular interactions mediating this regulation.

How can researchers implement multimodal single-cell analysis with At5g45510 antibody conjugates?

Implementing multimodal single-cell analysis with At5g45510 antibody conjugates requires careful optimization of several technical parameters:

Antibody conjugation optimization:

• Perform oligo-conjugation of At5g45510 antibodies using NHS-ester chemistry to attach DNA barcodes

• Test multiple antibody:oligo ratios (typically 1:10 to 1:30) to identify optimal conjugation conditions

• Purify conjugated antibodies using size exclusion chromatography

• Validate conjugate function using conventional flow cytometry against unconjugated antibody

Titration and panel design:

• Establish optimal working concentrations through serial dilution experiments (starting range between 0.05 and 10 μg/mL)

• In a 52-antibody panel, determine optimal At5g45510 antibody concentration based on epitope abundance and signal-to-noise ratio

• Test staining with various sample inputs: 10^6 cells in 50 μL antibody mixture for standard applications, scaling accordingly for limited samples

Single-cell protocol integration:

• Incorporate At5g45510 antibody conjugates into cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) protocols

• Implement feature barcoding technology for simultaneous detection of protein and mRNA

• Analyze data using dimensionality reduction and clustering algorithms to identify cell populations with distinct NRG1C expression patterns

• Correlate NRG1C protein levels with transcriptional profiles related to plant immunity

This approach enables unprecedented resolution in understanding how NRG1C expression varies at single-cell level and correlates with immune response heterogeneity across different cell types in plant tissues.

What are the challenges and solutions in developing cross-reactive At5g45510 antibodies for comparative studies across plant species?

Developing cross-reactive At5g45510 antibodies for comparative studies across plant species presents several challenges that require methodical solutions:

Challenge 1: Epitope conservation analysis

• Solution: Perform comprehensive sequence alignment of NRG1C homologs across target plant species

• Methodology: Use bioinformatic tools to identify conserved regions, particularly in functional domains

• Implementation: Design immunogens based on highly conserved epitopes within the NRG1 family

Challenge 2: Specificity versus cross-reactivity balance

• Solution: Generate monoclonal antibodies targeting multiple conserved epitopes

• Methodology: Immunize with synthetic peptides representing conserved regions

• Validation: Test specificity using recombinant proteins from multiple species and knockout controls

Challenge 3: Functional domain recognition

• Solution: Develop domain-specific antibodies targeting the truncated C-terminal region characteristic of NRG1C

• Methodology: Design antibodies that can distinguish NRG1C from full-length NRG1A/B based on structural differences

• Verification: Confirm domain specificity through competitive binding assays

Challenge 4: Post-translational modification interference

• Solution: Characterize species-specific post-translational modifications through mass spectrometry

• Methodology: Compare native protein samples with recombinant standards

• Application: Design epitope selection to minimize influence of variable modifications

Challenge 5: Validation across species

• Solution: Establish a comprehensive cross-species validation pipeline

• Methodology: Test antibodies against protein extracts from multiple plant species

• Verification: Implement knockout/knockdown controls for each species when available

These methodological approaches ensure that developed At5g45510 antibodies maintain both specificity for NRG1C versus other NLR family members and cross-reactivity across desired plant species for comparative immunological studies.

How should researchers troubleshoot inconsistent NRG1C detection in plant tissue samples?

When encountering inconsistent NRG1C detection in plant tissue samples, implement this systematic troubleshooting framework:

Problem: Weak or no signal

• Protein extraction efficiency:

  • Test alternative extraction buffers (add 0.1% SDS or 6M urea for membrane-associated proteins)

  • Implement mechanical disruption (bead beating) combined with chemical lysis

  • Use subcellular fractionation to enrich for compartments where NRG1C localizes

• Antibody factors:

  • Perform batch validation by ELISA against recombinant NRG1C protein

  • Test concentration range from 0.1-10 μg/mL to identify optimal antibody dilution

  • Confirm antibody storage conditions (avoid repeated freeze-thaw cycles)

• Epitope accessibility:

  • Test multiple antigen retrieval methods for fixed tissues

  • Evaluate alternative fixation protocols (paraformaldehyde vs. methanol)

  • Consider native vs. denaturing conditions for different applications

Problem: Non-specific binding

• Validation approaches:

  • Perform peptide competition assays to confirm specificity

  • Include nrg1c knockout negative controls in all experiments

  • Test pre-adsorption of antibody with related NLR proteins

• Protocol optimization:

  • Increase blocking reagent concentration (5-10% BSA or non-fat milk)

  • Extend blocking time (2-4 hours or overnight)

  • Add 0.1-0.5% Triton X-100 to reduce hydrophobic interactions

Problem: Variable results across tissues

• Expression level factors:

  • Quantify NRG1C transcript levels by RT-qPCR to correlate with protein detection

  • Consider tissue-specific expression patterns when interpreting negative results

  • Test samples at different developmental stages or after immune stimulation

• Analytical controls:

  • Include housekeeping protein controls for normalization

  • Implement biological replicates (minimum n=3) for each condition

  • Use recombinant protein standards for absolute quantification

How can researchers distinguish between NRG1C and other NLR family proteins in experimental analyses?

Distinguishing between NRG1C and other NLR family proteins, particularly its close relatives NRG1A and NRG1B, requires integrating multiple experimental approaches:

Antibody-based strategies:

• Epitope mapping: Develop antibodies targeting the unique C-terminal truncated region of NRG1C that differentiates it from full-length NRG1A/B

• Western blot analysis: Utilize molecular weight differences (NRG1C is smaller than full-length NRGs due to truncation) as primary discrimination method

• Immunoprecipitation-mass spectrometry: Pull down with broader specificity antibodies, then identify specific proteins through peptide sequencing

• Competition assays: Pre-incubate antibodies with recombinant NRG1A/B to demonstrate specificity for NRG1C

Complementary approaches:

• Gene-specific knockouts: Generate and validate nrg1c-specific knockout lines alongside nrg1a and nrg1b knockouts as controls

• Tagged protein expression: Express epitope-tagged versions (HA-tag, GFP) of each NRG protein for parallel detection

• Domain-specific functional assays: Exploit the antagonistic activity of NRG1C against NRG1A/B in functional competition assays

Functional discrimination:
When analyzing experimental data, researchers can distinguish NRG1C activity through its characteristic functional signatures:

• Autoimmunity suppression: NRG1C overexpression suppresses autoimmune phenotypes in chs3-2D and snc1 mutants

• Competition effects: NRG1C competes with NRG1A/B for shared signaling components without activating signaling

• Interaction profile: While sharing some interaction partners like SAG101 and EDS1, NRG1C has a distinct interactome that can be mapped through proximity labeling approaches

This multi-faceted approach ensures reliable discrimination between closely related NLR family members in complex experimental systems.

How might At5g45510 antibodies contribute to engineering disease-resistant crops?

At5g45510 antibodies provide essential tools for engineering disease-resistant crops through these methodological applications:

Precision breeding applications:

• Protein variant screening: Use At5g45510 antibodies to screen germplasm collections for natural NRG1C variants with enhanced regulatory function

• Transgenic validation: Quantify expression levels in genetically modified crops overexpressing optimized NRG1C variants

• Regulatory balance assessment: Monitor the ratio of NRG1C to NRG1A/B in engineered lines to maintain optimal immune homeostasis

Mechanistic insights for engineering:

• Structure-function mapping: Combine epitope-specific antibodies with mutational analysis to identify critical domains for NRG1C function

• Protein-protein interaction networks: Use co-immunoprecipitation with At5g45510 antibodies to map species-specific immune complexes

• Dynamics monitoring: Track NRG1C protein levels during infection to optimize expression timing in engineered systems

Crop improvement strategies:

• Balancing immunity and yield: Use antibody-based quantification to identify NRG1C expression levels that suppress autoimmunity without compromising pathogen resistance

• Multi-species implementation: Apply knowledge from Arabidopsis to engineer truncated NLR regulators in crop species using conserved principles

• Stacking with other resistance mechanisms: Develop multiplexed antibody panels to monitor multiple immune components simultaneously

The therapeutic potential of modulating NLR truncations like NRG1C represents a promising approach for developing crops with enhanced disease resistance while minimizing the fitness costs typically associated with constitutive immune activation.

What emerging technologies will enhance At5g45510 antibody development and applications?

Several emerging technologies will significantly advance At5g45510 antibody development and applications:

Next-generation antibody engineering:

• Single-domain antibodies: Develop nanobodies against NRG1C epitopes for enhanced tissue penetration and intracellular targeting

• AI-driven epitope prediction: Utilize machine learning algorithms to identify optimal immunogenic regions specific to NRG1C versus other NLRs

• Display technologies: Implement phage or yeast display for rapid screening of high-affinity antibody variants with enhanced specificity

Advanced detection platforms:

• Single-molecule imaging: Apply techniques like STORM or PALM microscopy with fluorescently-labeled At5g45510 antibodies to visualize individual NRG1C molecules in cellular contexts

• Mass cytometry (CyTOF): Develop metal-conjugated At5g45510 antibodies for high-dimensional analysis of plant immune responses

• Spatial transcriptomics integration: Combine At5g45510 antibody-based protein detection with spatial transcriptomic analysis for simultaneous protein-RNA mapping

Functional antibody developments:

• Intrabodies: Engineer cell-penetrating At5g45510 antibodies that can modulate NRG1C function within living plant cells

• Proximity-dependent labeling: Develop antibody-enzyme fusions (APEX2, TurboID) to map the NRG1C proximal proteome in different cellular contexts

• Optogenetic integration: Combine antibody-based detection with light-inducible systems to enable temporal control of NRG1C activity visualization

High-throughput validation systems:

• Antibody validation arrays: Develop protein arrays containing multiple NLR family members for rapid specificity testing

• Automated quality control: Implement standardized workflows with quantitative metrics for batch consistency evaluation

• Digital-PCR-coupled immunoassays: Combine antibody capture with digital PCR for absolute quantification of NRG1C proteins

These technological advances will enable unprecedented insights into NRG1C biology and facilitate the development of more effective plant immune engineering strategies.