At5g45440 Antibody

What is the function of At5g45440 in plant immunity pathways?

At5g45440 encodes a nucleotide-binding leucine-rich repeat (NLR) protein that functions within the plant immune signaling network. Similar to other NLRs like NRG1C, it likely plays a role in TNL (TIR-NBS-LRR)-mediated immunity pathways. Research indicates that NLR proteins can have both positive and negative regulatory roles in plant immunity, depending on their expression levels and interactions with other immune components . Methodologically, researchers should consider analyzing expression levels in various immune mutants and under pathogen challenge conditions, as demonstrated with NRG1C, which showed dramatic upregulation (200-7,000 fold) in autoimmune mutants like snc1 and chs3-2D .

What detection methods are most effective for At5g45440 antibody-based experiments?

For reliable detection of At5g45440 protein, researchers should employ multiple complementary techniques:

• Western blotting: Use reducing conditions with appropriate immunoblot buffers, similar to protocols established for other plant immune proteins. Optimization of primary antibody concentration (typically 1-5 μg/mL) is essential for specific detection .

• Immunofluorescence: Immersion fixation followed by incubation with the primary antibody (20-30 μg/mL) for 3-4 hours at room temperature, then visualization with fluorophore-conjugated secondary antibodies can effectively localize the protein in plant cells .

• ELISA: Direct ELISA approaches can quantify protein levels when standardized against known concentrations .

When developing detection protocols, researchers should validate antibody specificity using knockout mutants as negative controls and include appropriate loading controls for quantitative analyses.

How should At5g45440 antibodies be stored to maintain optimal activity?

For maximum retention of antibody activity:

• Store lyophilized antibodies at -20°C to -80°C until reconstitution

• After reconstitution in sterile PBS or similar buffer, store working aliquots at 4°C for short-term use (1-2 weeks)

• For long-term storage, prepare small aliquots to minimize freeze-thaw cycles and store at -20°C

• Add carrier proteins (0.1-1% BSA) to diluted antibodies to prevent adsorption to storage tubes

• Monitor antibody performance regularly with positive controls to detect any deterioration in activity

Empirical testing shows that properly stored antibodies typically maintain >90% of their activity for at least 12 months when stored as recommended.

How can researchers optimize immunoprecipitation protocols for studying At5g45440 protein interactions?

Optimizing immunoprecipitation (IP) protocols for At5g45440 requires careful consideration of experimental conditions:

• Extraction buffer optimization: Test multiple buffer compositions varying in ionic strength (150-500 mM NaCl), detergent type (0.1-1% NP-40, Triton X-100, or CHAPS), and pH (6.8-8.0) to maximize protein solubility while preserving interactions.

• Cross-linking considerations: For transient interactions, implement formaldehyde (0.5-2%) or DSP (dithiobis(succinimidyl propionate)) cross-linking prior to cell lysis.

• Antibody coupling strategies: Compare results between traditional methods using Protein A/G beads and direct covalent coupling to activated beads to reduce background.

• Negative controls: Always include both IgG isotype controls and samples from knockout plants lacking At5g45440 to identify non-specific interactions.

Similar to studies with NRG1C, researchers should particularly investigate interactions with other immunity-related proteins that function in TNL-mediated pathways, including potential associations with SAG101 and other NRG1 family members that show overlapping phenotypes .

What strategies can address epitope masking issues in At5g45440 immunodetection experiments?

Epitope masking can significantly impact At5g45440 detection, particularly when protein-protein interactions occur in immunity complexes. To overcome this challenge:

• Sample preparation modifications:

  • Test multiple denaturing conditions (heat, SDS, urea)

  • Evaluate non-reducing vs. reducing conditions

  • Explore gentle detergent solubilization protocols

• Epitope retrieval techniques:

  • For fixed tissues, implement heat-induced epitope retrieval (HIER) protocols (80-95°C in citrate buffer, pH 6.0)

  • Test enzymatic treatments with proteases for masked epitopes

  • Consider sonication to improve antibody accessibility

• Multiple antibody approach:

  • Use antibodies targeting different epitopes within At5g45440

  • Combine N-terminal and C-terminal specific antibodies to confirm results

In immunity studies, protein conformational changes upon activation may hide epitopes. Similar to observations with NRG1C, where protein function changes in different genetic backgrounds , researchers should validate detection methods across multiple experimental conditions.

How should researchers quantify At5g45440 expression changes during pathogen infection?

For accurate quantification of At5g45440 expression changes during pathogen challenge:

When performing these analyses, include multiple time points post-infection (early: 0-6h, middle: 12-24h, late: 48-72h) to capture the full expression dynamics. Similar to NRG1C, which shows dramatic upregulation upon pathogen infection or in autoimmune mutants , At5g45440 expression should be monitored in both wild-type plants and immune-related mutants to understand its regulation in different genetic contexts.

How can CRISPR-engineered plants help validate At5g45440 antibody specificity?

CRISPR-engineered plant lines provide essential tools for validating antibody specificity:

• Complete knockout validation:

  • Generate complete gene deletions using paired sgRNAs

  • Confirm absence of signal in Western blot, immunohistochemistry, and IP experiments

  • These lines serve as gold-standard negative controls

• Epitope-modified variants:

  • Engineer specific mutations in the antibody recognition site

  • Create small deletions or substitutions at the epitope region

  • Use these lines to confirm epitope-specific binding

• Tagged variant lines:

  • Create C- or N-terminal tagged versions at the endogenous locus

  • Compare antibody detection with tag-based detection

  • Correlation between signals confirms specificity

• Truncation series:

  • Generate plants expressing truncated variants

  • Map the exact binding region of the antibody

  • Particularly important for antibodies against N-terminal regions, as seen with truncated NLRs like NRG1C

This approach has successfully validated other plant immune receptor antibodies and provides definitive evidence for specificity beyond traditional Western blot analyses.

What are the best approaches for studying post-translational modifications of At5g45440?

Post-translational modifications (PTMs) significantly impact plant immune protein function. For comprehensive PTM analysis of At5g45440:

• Phosphorylation profiling:

  • Immunoprecipitate At5g45440 using validated antibodies

  • Perform phospho-enrichment using TiO₂ or IMAC

  • Analyze by LC-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD)

  • Compare phosphorylation patterns before and after pathogen challenge

• Ubiquitination detection:

  • Use tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

  • Immunoprecipitate At5g45440 and probe with anti-ubiquitin antibodies

  • Alternatively, express His-tagged ubiquitin and perform Ni-NTA pulldowns

  • Identify ubiquitination sites by mass spectrometry

• Glycosylation analysis:

  • Treat immunoprecipitated protein with PNGase F or other glycosidases

  • Observe mobility shifts by Western blot

  • For detailed glycan profiling, employ specialized mass spectrometry techniques

  • Consider specific glycosylation inhibitors in functional studies

• SUMOylation assessment:

  • Use SUMO-specific antibodies in co-IP experiments

  • Express tagged SUMO constructs for pulldown assays

  • Implement site-directed mutagenesis of predicted SUMOylation sites

Similar to other plant NLRs, At5g45440 likely undergoes multiple PTMs that regulate its stability, localization, and signaling capacity during immune responses.

How can researchers differentiate between specific and non-specific binding in At5g45440 immunoprecipitation experiments?

Distinguishing specific from non-specific binding requires rigorous experimental controls and validation steps:

• Comprehensive control panel:

  • IgG isotype controls matched to the antibody species and subclass

  • Pre-immune serum controls when available

  • Knockout/knockdown plant samples lacking At5g45440

  • Competition with excess antigenic peptide

• Stringency optimization:

  • Perform parallel IPs with increasing salt concentrations (150mM to 500mM)

  • Test multiple detergent types and concentrations

  • Plot persistence curves for each interactor across stringency conditions

  • True interactors typically remain bound at higher stringency

• Crosslinking validation:

  • Compare results with and without crosslinking

  • Use membrane-permeable crosslinkers for in vivo interactions

  • Employ proximity labeling techniques (BioID or APEX) as orthogonal validation

• Reciprocal IP confirmation:

  • For putative interactors, perform reverse IPs

  • Confirmation in both directions significantly increases confidence

  • Quantify interaction stoichiometry when possible

When analyzing interaction candidates, researchers should be particularly attentive to proteins involved in immune complex formation, as At5g45440 may participate in signaling networks similar to those involving NRG1C .

How can researchers address inconsistent results in At5g45440 detection across different plant tissues?

Inconsistent detection across tissues often stems from tissue-specific variables that require methodological adjustments:

• Tissue-specific extraction optimization:

  • Adjust buffer compositions based on tissue type (leaves, roots, flowers)

  • For tissues with high phenolic content, add PVPP (2-5%) and increased antioxidants

  • For tissues with high lipid content, increase detergent concentrations

  • Consider tissue-specific protease inhibitor cocktails

• Fixation and embedding protocols:

  • Compare crosslinking fixatives (formaldehyde, glutaraldehyde) with precipitating fixatives (acetone, methanol)

  • Optimize fixation times for each tissue type

  • Test multiple embedding media if performing immunohistochemistry

• Signal amplification strategies:

  • Implement tyramide signal amplification for low-abundance detection

  • Use biotin-streptavidin systems for enhanced sensitivity

  • Consider dual antibody detection systems

• Expression level normalization:

  • Develop tissue-specific loading controls

  • Implement absolute quantification using recombinant protein standards

  • Consider digital droplet PCR for transcript level normalization

Researchers should systematically document At5g45440 detection parameters across different tissues to build a comprehensive methodological framework, similar to approaches used for characterizing expression patterns of other plant immunity proteins .

What bioinformatic approaches can predict potential cross-reactivity of At5g45440 antibodies?

To predict and mitigate potential cross-reactivity of At5g45440 antibodies:

• Epitope mapping and homology analysis:

  • Determine the exact epitope sequence recognized by the antibody

  • Perform BLAST searches against the entire plant proteome

  • Identify proteins with similar epitope sequences

  • Focus particularly on related NLR family members that may share structural features

• Structural prediction approaches:

  • Generate 3D structural models of At5g45440 and related proteins

  • Compare surface-exposed regions that could serve as antibody epitopes

  • Use molecular dynamics simulations to assess epitope accessibility

• Machine learning prediction tools:

  • Implement algorithms trained on antibody-epitope interaction data

  • Use these to predict possible cross-reactive proteins

  • Balance sensitivity and specificity in prediction parameters

• Experimental validation pipeline:

  • Express recombinant versions of predicted cross-reactive proteins

  • Test antibody binding through direct ELISA or Western blot

  • Quantify relative binding affinities to each protein

These approaches are particularly important when studying At5g45440 in the context of other NLR family members, as structural similarities can lead to unexpected cross-reactivity, similar to challenges encountered with antibodies against other plant immune proteins .

How should researchers interpret contradictory results between transcript and protein levels of At5g45440?

Discrepancies between transcript and protein measurements are common in plant immunity studies and require careful interpretation:

• Mechanisms explaining transcript-protein discordance:

  • Post-transcriptional regulation via miRNAs or RNA-binding proteins

  • Altered protein stability or degradation rates during immune responses

  • Translational efficiency changes under stress conditions

  • Protein compartmentalization or sequestration affecting extraction efficiency

• Validation approaches:

  • Pulse-chase experiments to determine protein half-life

  • Polysome profiling to assess translation efficiency

  • Proteasome inhibitor treatments to evaluate degradation contributions

  • Comparison across multiple time points to detect temporal disconnects

• Integrated analysis framework:

  • Correlate transcript, protein, and functional phenotypes

  • Develop mathematical models to account for time delays

  • Consider both absolute levels and rates of change

  • Implement time-course experiments with high temporal resolution

• Experimental design considerations:

  • Include both unchallenged and pathogen-challenged samples

  • Compare results across different genetic backgrounds

  • Test multiple extraction methods to ensure complete protein recovery

Similar to observations with NRG1C, where overexpression yielded unexpected phenotypic outcomes , At5g45440 protein levels may not directly correlate with transcript abundance, particularly during dynamic immune responses.

What emerging technologies will advance At5g45440 antibody-based research?

Several cutting-edge technologies hold promise for enhancing At5g45440 research:

• Single-cell antibody-based techniques:

  • Adaptation of CyTOF for plant single-cell analysis

  • Single-cell Western blotting for heterogeneity assessment

  • Microfluidic antibody-based sorting of plant protoplasts

  • These approaches will reveal cell-specific expression patterns

• Advanced microscopy applications:

  • Super-resolution microscopy (STORM, PALM) for nanoscale localization

  • Expansion microscopy to physically magnify subcellular structures

  • Lattice light-sheet microscopy for long-term live imaging

  • These methods will clarify the dynamic subcellular localization of At5g45440

• Proximity labeling advances:

  • TurboID and miniTurbo for rapid biotin labeling of proximal proteins

  • Split-BioID for detecting specific protein-protein interactions

  • APEX2 for spatially restricted labeling

  • These techniques will map the dynamic At5g45440 interactome

• Antibody engineering approaches:

  • Nanobody development for improved intracellular tracking

  • Bispecific antibodies to detect protein complexes in situ

  • Antibody fragments with enhanced tissue penetration

  • These tools will expand the repertoire of immunodetection options

These technologies will particularly benefit studies of plant immune receptors by enabling more precise spatial and temporal resolution of signaling events, similar to advances being made in the broader field of plant-pathogen interactions .

How might synthetic biology approaches enhance At5g45440 antibody production and specificity?

Synthetic biology offers innovative solutions to antibody production challenges:

• Plant-based expression systems:

  • Transient expression in Nicotiana benthamiana

  • Stable transgenic lines in Arabidopsis or tobacco

  • Chloroplast transformation for high-yield production

  • These systems can achieve yields of 1-2 mg antibody per gram fresh weight

• Modular antibody design:

  • Synthetic scaffolds combining multiple binding domains

  • Orthogonal epitope tagging for multiplexed detection

  • Programmable binding domains with tunable affinity

  • These approaches enable customized detection reagents

• Computationally designed epitopes:

  • In silico identification of highly specific regions

  • Structure-based epitope optimization

  • Machine learning algorithms to predict immunogenicity

  • These methods improve specificity and reduce cross-reactivity

• Cell-free production systems:

  • Wheat germ extract for plant-compatible folding

  • Microfluidic-based continuous synthesis platforms

  • Ribosome display for rapid selection of high-affinity variants

  • These technologies enable rapid prototype testing

Implementing these synthetic biology approaches could significantly enhance the specificity and utility of At5g45440 antibodies, particularly when studying complex immune signaling networks that involve numerous structurally related proteins .