At1g15890 Antibody

What is the At1g15890 protein and why is it significant in research?

At1g15890 is a probable disease resistance protein first identified in Arabidopsis thaliana and also found in other plant species like Beta vulgaris subsp. vulgaris (sugar beet) . It belongs to the nucleotide-binding-leucine-rich repeat (NLR) family of proteins that play crucial roles in plant immune systems . These resistance proteins function as immune receptors that recognize pathogen effectors and trigger defense responses. Research on At1g15890 is significant for understanding fundamental plant immunity mechanisms, potentially leading to improved crop disease resistance strategies.

What experimental approaches are most effective for studying At1g15890 expression patterns?

For studying At1g15890 expression patterns, a multi-method approach yields the most comprehensive results:

• Quantitative RT-PCR: Enables precise quantification of At1g15890 transcript levels under various conditions

• Microarray analysis: Provides broader context by allowing comparison of At1g15890 expression with other genes

• RNA-Seq: Offers high sensitivity for detecting expression changes across different tissues and treatments

• GFP fusion proteins: Allow visualization of protein localization and dynamics in living cells

When designing experiments, consider that expression of resistance genes like At1g15890 can vary significantly based on developmental stage, tissue type, and stress conditions. Analysis should include appropriate reference genes and biological replicates (minimum n=3) for statistical validation .

How does At1g15890 interact with other disease resistance pathways in plants?

At1g15890 functions within a complex network of disease resistance mechanisms. Research indicates that NLR proteins like At1g15890 can interact with:

• Signaling hubs: May associate with helper NLRs like ADR1 homologs (ADR1, ADR1-L1, ADR1-L2) or NRG1 homologs that transduce defense signals

• Actin cytoskeleton components: Evidence suggests connection with actin depolymerizing factors (ADFs), which affect nuclear organization and subsequently gene expression of resistance pathways

• Transcriptional networks: Influences expression of other defense-related genes as demonstrated by microarray analyses of ADF mutants, which showed altered expression of multiple NLR genes

These interactions create a sophisticated surveillance system that allows plants to recognize diverse pathogens and mount appropriate defense responses. Research approaches combining co-immunoprecipitation with mass spectrometry can help elucidate these protein-protein interactions.

What are the optimal antigen design strategies for generating specific At1g15890 antibodies?

Generating highly specific antibodies against At1g15890 requires careful antigen design:

• Epitope selection considerations:

  • Choose unique regions that differ from homologous NLR proteins

  • Target accessible regions on the protein surface

  • Avoid transmembrane domains and highly conserved functional domains

  • Consider both N-terminal (CC domain) and C-terminal (LRR region) epitopes for comprehensive coverage

• Recommended approaches:

  • Synthetic peptides (15-20 amino acids) conjugated to carrier proteins

  • Recombinant protein fragments expressed in E. coli (preferably the variable regions)

  • Full-length protein expression using plant-based systems for proper folding

The coiled-coil (CC) domain of At1g15890 shows distinct structural features compared to canonical CNLs, with three C-terminal α-helices predicted to form coiled-coils rather than the first α-helix . This structural uniqueness can be leveraged for antibody specificity.

What validation methods ensure specificity when working with At1g15890 antibodies?

A comprehensive validation strategy includes:

For maximum confidence, perform validation across multiple plant tissues and under various stress conditions, as NLR expression can be highly context-dependent .

How do I troubleshoot cross-reactivity issues with At1g15890 antibodies?

Cross-reactivity with other NLR proteins is a common challenge when working with At1g15890 antibodies. Systematic troubleshooting approaches include:

• Identify cross-reacting proteins:

  • Perform mass spectrometry on immunoprecipitated samples

  • Compare western blot patterns across tissues with different NLR expression profiles

  • Test against recombinant proteins from closely related NLR families

• Optimization strategies:

  • Increase washing stringency with higher salt concentrations (300-500 mM NaCl)

  • Add low concentrations of competitive agents (0.1-0.5% Tween-20)

  • Perform antibody pre-absorption with recombinant proteins from cross-reacting NLRs

  • Consider epitope-tagged versions of At1g15890 for specific applications

• Application-specific recommendations:

  • For western blots: Optimize primary antibody dilution (typically 1:500-1:2000)

  • For immunoprecipitation: Use crosslinking to stabilize transient interactions

  • For immunofluorescence: Include additional blocking agents (5% BSA, 5% normal serum)

Remember that complete elimination of cross-reactivity may not be achievable for highly conserved protein families. Multiple antibodies targeting different epitopes can provide validation through concordant results.

What immunoprecipitation protocols are most effective for studying At1g15890 protein interactions?

For investigating At1g15890 protein interactions, consider these optimized immunoprecipitation (IP) approaches:

• Native IP protocol:

  • Extract proteins in mild buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, protease inhibitors)

  • Pre-clear lysate with protein A/G beads (1 hour, 4°C)

  • Incubate with At1g15890 antibody overnight at 4°C (5-10 μg antibody per mg protein)

  • Capture with protein A/G beads (2-3 hours, 4°C)

  • Wash 4-5 times with buffer containing 0.1% NP-40

  • Elute with 0.1 M glycine pH 2.5 or SDS loading buffer

• Crosslinking IP for transient interactions:

  • Treat intact tissues with 1-2% formaldehyde (10-15 minutes)

  • Quench with 125 mM glycine

  • Extract and immunoprecipitate as above

  • Reverse crosslinks (95°C, 10 minutes in SDS buffer) before analysis

• Proximity-dependent approaches:

  • Express At1g15890 fused to BioID or TurboID biotin ligase

  • Supply biotin to living cells (50 μM, 1-3 hours)

  • Capture biotinylated proximal proteins with streptavidin

  • Identify by mass spectrometry

Combining these approaches can reveal both stable and transient interactions of At1g15890 with other proteins in the plant immune signaling pathway .

How can At1g15890 antibodies be used to study chromatin dynamics during immune responses?

Recent research suggests connections between NLR proteins, nuclear organization, and gene expression regulation . At1g15890 antibodies can be employed to study these processes through:

• Chromatin immunoprecipitation (ChIP):

  • Fix plant tissue with 1% formaldehyde (10 minutes)

  • Isolate nuclei and fragment chromatin (sonication to 200-500 bp fragments)

  • Immunoprecipitate with At1g15890 antibodies

  • Analyze by qPCR or sequencing to identify genomic binding regions

• Immunofluorescence microscopy:

  • Fix and permeabilize plant cells

  • Co-stain with At1g15890 antibodies and DNA/chromatin markers

  • Analyze changes in nuclear localization during immune activation

  • Quantify parameters such as chromocenter size and nuclear distribution

• Proximity ligation assay (PLA):

  • Co-stain with At1g15890 antibody and antibodies against chromatin-modifying enzymes

  • Apply species-specific secondary antibodies with DNA probes

  • Amplify signal when proteins are within 40 nm

  • Quantify interaction events per nucleus

Studies with actin depolymerizing factors (ADFs) have demonstrated that cytoskeletal components can influence chromocenter size and nuclear organization, with subsequent effects on disease resistance gene expression . Similar approaches could reveal At1g15890's role in this process.

What considerations are important when using At1g15890 antibodies for tissue immunohistochemistry?

Successful immunohistochemistry with At1g15890 antibodies requires:

• Tissue preparation options:

  • Paraffin embedding: Provides excellent morphology but may compromise antigen accessibility

  • Cryo-sectioning: Better antigen preservation but more challenging morphology

  • Whole-mount: Allows 3D visualization but limited to thin tissues or organs

• Antigen retrieval methods:

  • Heat-induced: 10 mM sodium citrate buffer (pH 6.0), 95°C for 20 minutes

  • Enzymatic: Proteinase K (10 μg/ml, 10 minutes at room temperature)

  • Test multiple methods as NLR proteins can be sensitive to fixation

• Signal detection systems:

  • Chromogenic: DAB precipitation for permanent samples and light microscopy

  • Fluorescent: Multiple fluorophores for co-localization studies

  • Tyramide signal amplification: For low-abundance targets

• Controls and quantification:

  • Include both positive tissues (known to express At1g15890) and negative controls

  • Use knockout/knockdown tissues as gold-standard negative controls

  • Implement automated image analysis for objective quantification

Expression analysis has shown that NLR genes like At1g15890 can have tissue-specific expression patterns and can be induced under certain stress conditions . Experimental design should account for this variability.

How can At1g15890 antibodies contribute to understanding plant immune signaling dynamics?

At1g15890 antibodies enable sophisticated studies of immune signaling dynamics:

• Temporal signaling studies:

  • Use time-course experiments to track At1g15890 modifications (phosphorylation, ubiquitination)

  • Combine with phospho-specific antibodies to map activation sequences

  • Monitor protein complex formation at different stages of immune response

• Spatial signaling analysis:

  • Track At1g15890 translocation between cellular compartments

  • Investigate membrane association during immune activation

  • Analyze nuclear accumulation and potential DNA interactions

• Quantitative approaches:

  • Implement pulse-chase immunoprecipitation to measure protein turnover rates

  • Use antibody-based biosensors for real-time monitoring in live cells

  • Apply super-resolution microscopy to visualize nanoscale distribution

Research suggests that plant NLR proteins function in preformed complexes that undergo conformational changes upon pathogen detection . At1g15890 antibodies can help elucidate these structural rearrangements and subsequent signaling events.

What approaches can reconcile contradictory findings regarding At1g15890 function across different experimental systems?

Researchers often encounter contradictory findings when studying complex proteins like At1g15890. Reconciliation approaches include:

• Systematic comparison of experimental conditions:

  • Document all variables: plant age, tissue type, growth conditions, pathogen strains

  • Create standardized reference samples for cross-laboratory validation

  • Implement meta-analysis techniques to identify pattern-driving variables

• Antibody-based functional studies:

  • Use epitope-specific antibodies to block distinct protein domains

  • Apply antibodies to isolated subcellular fractions to test compartment-specific functions

  • Compare results with genetic approaches (knockout, knockdown, overexpression)

• Integrative multi-omics approaches:

  • Combine antibody-based proteomics with transcriptomics and metabolomics

  • Apply network modeling to contextualize contradictory results

  • Identify conditional factors that explain discrepancies

Studies on related NLR proteins have shown that their functions can vary dramatically depending on genetic background and environmental conditions . For instance, expression of some NLR genes is significantly altered in actin depolymerizing factor mutants, suggesting complex regulatory networks that may explain seemingly contradictory results.

How can single-cell approaches with At1g15890 antibodies reveal cellular heterogeneity in immune responses?

Emerging single-cell technologies with At1g15890 antibodies can uncover immune response heterogeneity:

• Single-cell antibody-based methods:

  • Flow cytometry with At1g15890 antibodies to identify responsive cell populations

  • Mass cytometry (CyTOF) incorporating metal-tagged antibodies for multiparameter analysis

  • Imaging mass cytometry for spatial single-cell protein profiling in tissue context

• Integrative single-cell approaches:

  • CITE-seq combining At1g15890 antibody detection with single-cell RNA-seq

  • Spatial transcriptomics with antibody validation of key markers

  • Single-cell Western blotting for protein isoform analysis

• Microfluidic applications:

  • Droplet-based single-cell immunoassays for At1g15890 quantification

  • Microwell arrays for monitoring single-cell secretion dynamics

  • Organ-on-chip approaches for controlled microenvironment studies

Single-cell methodologies can help identify specialized "sentinel" cells that may initiate defense responses. Recent research in paired antibody repertoire sequencing demonstrates how single-cell approaches can provide insights into adaptive immune responses that would be masked in bulk analyses .

How might CRISPR/Cas9 epitope tagging enhance At1g15890 antibody applications?

CRISPR/Cas9 epitope tagging offers transformative opportunities for At1g15890 research:

• Endogenous tagging advantages:

  • Maintains native gene regulation and expression levels

  • Avoids artifacts associated with overexpression systems

  • Enables tracking of all protein isoforms from the endogenous locus

• Strategic tagging approaches:

  • C-terminal tags for proteins where N-terminus contains critical domains

  • Internal tags within flexible linker regions for proteins sensitive to terminal modifications

  • Split-tag complementation for studying protein-protein interactions

• Advanced applications with tagged lines:

  • Chromatin profiling through CUT&Tag rather than traditional ChIP

  • Proximity labeling with TurboID fusions for interactome mapping

  • Live-cell single-molecule tracking with Halo or SNAP tags

Current techniques like CloneEZ™ Seamless cloning technology can facilitate the generation of these constructs . Tagged lines can then serve as positive controls for antibody validation and enable applications where current antibodies have limitations.

What role might At1g15890 antibodies play in understanding epigenetic regulation of plant immunity?

At1g15890 antibodies can facilitate research into epigenetic dimensions of plant immunity:

• Chromatin-associated functions:

  • Investigate potential association with specific chromatin states

  • Analyze co-localization with chromatin modifiers during defense responses

  • Assess impact on DNA methylation patterns at defense gene loci

• Nuclear organization studies:

  • Examine relationship between At1g15890 and chromocenter dynamics

  • Investigate associations with nuclear bodies during immune responses

  • Analyze changes in chromosome territories during resistance responses

• Methodological approaches:

  • ChIP-seq with At1g15890 antibodies followed by epigenetic mark analysis

  • Re-ChIP to identify co-occurrence with specific histone modifications

  • Proximity ligation assays to detect interactions with epigenetic machinery

Research has revealed connections between actin depolymerizing factors, nuclear morphology, and expression of NLR genes including potentially At1g15890 . Chromocenters (condensed heterochromatin regions) show altered size and distribution in mutants with disturbed actin, suggesting a potential link between cytoskeletal organization, nuclear architecture, and disease resistance gene regulation that could be further explored using At1g15890 antibodies.

How can computational approaches improve At1g15890 antibody design and application?

Computational methods enhance At1g15890 antibody research in multiple dimensions:

• Structure-based antibody design:

  • Predict epitope accessibility through protein structure modeling

  • Identify surface regions unique to At1g15890 versus other NLRs

  • Simulate antibody-antigen interactions to optimize binding affinity

• Bioinformatic analysis of experimental data:

  • Implement machine learning for automated western blot/IF quantification

  • Apply network analysis to interpret immunoprecipitation-mass spectrometry results

  • Develop algorithms for single-cell immune response profiling

• Integrated data analysis frameworks:

  • Create unified databases of antibody validation results across laboratories

  • Develop standardized pipelines for antibody specificity assessment

  • Implement FAIR (Findable, Accessible, Interoperable, Reusable) principles for antibody data

Computational approaches can also help identify polymorphisms in At1g15890 across plant varieties and predict how these might affect antibody binding, similar to approaches used in studying genetic polymorphisms affecting antibody production in human vaccination studies .