At2g40995 Antibody

What is the At2g40995 gene in Arabidopsis thaliana and what protein does it encode?

At2g40995 is a gene in Arabidopsis thaliana (Mouse-ear cress) that encodes a defensin-like protein 107 belonging to the molecular chaperone Hsp40/DnaJ family protein . Defensin-like proteins typically function in plant defense mechanisms, while Hsp40/DnaJ family proteins serve as molecular chaperones that assist in protein folding, assembly, and translocation processes. Current research suggests potential roles for At2g40995 in stress response pathways, though specific functions need further characterization through targeted experimental approaches.

What are the primary applications of At2g40995 antibody in plant research?

The primary research applications for At2g40995 antibody include:

These techniques allow researchers to investigate At2g40995 protein expression, localization, and interactions within plant systems, providing essential insights into its biological functions.

What are the characteristics of commercially available At2g40995 antibodies?

Currently available At2g40995 antibodies have the following specifications:

These characteristics should be considered when selecting an appropriate antibody for specific experimental designs and research questions.

How should researchers validate the specificity of At2g40995 antibody?

Proper validation is critical for ensuring reliable experimental results:

• Western Blot Validation:

  • Compare wild-type Arabidopsis samples with At2g40995 knockout/knockdown lines

  • Verify single band at the expected molecular weight

  • Test for cross-reactivity with related defensin-like or Hsp40/DnaJ family proteins

• Peptide Competition Assay:

  • Pre-incubate antibody with the immunizing peptide

  • Compare signal with and without peptide competition

  • Significant signal reduction confirms specificity

• Multiple Antibody Validation:

  • Use antibodies targeting different epitopes of At2g40995

  • Compare detection patterns across experimental conditions

• Recombinant Protein Controls:

  • Test against purified recombinant At2g40995

  • Include related proteins as negative controls

What are best practices for storage and handling of At2g40995 antibodies?

To maintain antibody functionality:

Proper storage and handling practices maintain antibody specificity and sensitivity, ensuring consistent experimental results across studies.

How can At2g40995 antibody be integrated into studies of drought adaptation in Arabidopsis?

Recent research on genotype-environment associations (GEA) in Arabidopsis provides a framework for investigating At2g40995's potential role in drought adaptation:

• Protein Expression Profiling:

  • Compare At2g40995 protein expression between drought-sensitive and drought-resistant ecotypes

  • Analyze expression patterns during progressive drought stress

  • Correlate protein levels with physiological drought response markers

• Protein Interaction Networks:

  • Use At2g40995 antibody for co-immunoprecipitation followed by mass spectrometry

  • Identify drought-specific interaction partners

  • Map protein complexes formed under drought stress conditions

• Integration with GEA Data:

  • Correlate At2g40995 protein levels with allelic variants identified in GEA studies

  • Examine protein expression in the context of genotype-by-environment (GxE) interactions

  • Assess post-translational modifications in response to moisture gradients

These approaches can help determine whether At2g40995, like the drought-responsive genes WRKY38 and LSD1 identified in recent studies, contributes to adaptive drought responses in Arabidopsis .

What methodological approaches can improve the specificity of At2g40995 antibodies through antibody phage display technology?

Antibody phage display offers powerful approaches for developing highly specific At2g40995 antibodies:

• Library Construction Strategy:

  • Create antibody libraries from immunized rabbits or synthetic repertoires

  • Amplify VH and VL sequences using degenerate primers spanning all variable region families

  • Construct scFv (single-chain variable fragment) libraries with ≥10⁶ diversity

• Selection (Panning) Protocol:

  • Immobilize recombinant At2g40995 protein on solid support

  • Perform 3-4 rounds of selection with increasing stringency

  • Include negative selection steps with related proteins to enhance specificity

• Clone Screening and Validation:

  • Analyze monoclonal phage by ELISA against At2g40995 and related proteins

  • Sequence promising clones to determine VH and VL sequences

  • Express soluble scFv for functional characterization

Recent advances in antibody design have demonstrated that precise, specific antibodies can be developed without prior antibody information, using yeast display scFv libraries of approximately 10⁶ sequences .

How can researchers design experiments to distinguish between At2g40995 and other defensin-like proteins?

Distinguishing At2g40995 from related proteins requires careful experimental design:

• Epitope Mapping:

  • Identify unique regions in At2g40995 compared to other defensin-like proteins

  • Design peptide arrays covering the entire At2g40995 sequence

  • Map the specific epitopes recognized by the antibody

• Cross-Reactivity Testing:

  • Express and purify recombinant At2g40995 and related defensin-like proteins

  • Perform systematic Western blot and ELISA testing across all family members

  • Quantify relative binding affinities to each protein

• Genetic Validation:

  • Use At2g40995 knockout/knockdown lines as negative controls

  • Compare antibody reactivity in wild-type vs. mutant tissues

  • Perform complementation with tagged At2g40995 variants

• Competitive Binding Assays:

  • Pre-incubate antibodies with purified At2g40995 or related proteins

  • Compare signal reduction patterns to quantify cross-reactivity

  • Develop absorption protocols to enhance specificity

What analytical frameworks best integrate At2g40995 protein data with genetic and environmental studies?

Integrating protein-level data with genetic and environmental analyses requires sophisticated analytical approaches:

• Multi-Omics Data Integration:

  • Correlate At2g40995 protein levels with transcriptomic and metabolomic data

  • Implement network analysis to identify functional modules

  • Use machine learning to predict environmental response patterns

• Statistical Methods for GxE Analysis:

  • Apply mixed linear models integrating protein expression and genetic variation

  • Use structural equation modeling to map causal relationships

  • Implement Bayesian approaches to estimate effect sizes

Recent GEA studies in Arabidopsis have successfully integrated genetic and environmental data to identify drought-responsive genes (e.g., WRKY38, LSD1), providing a methodological framework for studying At2g40995 .

How can researchers use At2g40995 antibody to investigate post-translational modifications during stress responses?

Investigating stress-induced post-translational modifications (PTMs) of At2g40995:

• Modification-Specific Detection:

  • Develop or obtain antibodies specific to common PTMs (phosphorylation, ubiquitination)

  • Compare modified vs. total At2g40995 levels under stress conditions

  • Map modification sites using mass spectrometry

• Subcellular Dynamics:

  • Track At2g40995 localization changes during stress response

  • Correlate localization with modification status

  • Examine co-localization with known stress response components

• Functional Significance Assessment:

  • Express wild-type vs. modification-site mutant versions of At2g40995

  • Compare protein-protein interactions of modified vs. unmodified protein

  • Assess impact of modifications on protein stability and turnover

• Temporal Profiling:

  • Create detailed time-course analyses of modifications during stress progression

  • Correlate modification patterns with physiological stress markers

  • Identify regulatory enzymes controlling At2g40995 modifications

These approaches can reveal how post-translational regulation of At2g40995 contributes to plant stress responses, particularly in drought adaptation contexts where genotype-by-environment interactions have been observed .

What are the key considerations when designing immunoprecipitation experiments with At2g40995 antibody?

Successful immunoprecipitation requires attention to several critical factors:

• Sample Preparation:

  • Use optimized extraction buffers compatible with plant tissues

  • Include protease/phosphatase inhibitors to preserve protein modifications

  • Consider native vs. denaturing conditions based on experimental goals

• Antibody Selection and Coupling:

  • Evaluate polyclonal vs. monoclonal antibodies for IP efficiency

  • Consider direct antibody labeling vs. protein A/G beads

  • Optimize antibody-to-lysate ratios through titration experiments

• Control Implementation:

  • Include non-immune IgG controls from the same species

  • Use At2g40995 knockout/knockdown samples as negative controls

  • Consider pre-clearing lysates to reduce non-specific binding

• Validation Approaches:

  • Confirm successful IP by Western blot of input, unbound, and eluted fractions

  • Verify enrichment of At2g40995 by quantitative comparison to input

  • Validate interacting partners through reciprocal co-IP experiments

These methodological considerations help ensure reliable and reproducible immunoprecipitation results when studying At2g40995 protein interactions.

How can antibody phage display be optimized for generating high-affinity At2g40995-specific antibodies?

Optimizing antibody phage display for At2g40995-specific antibodies:

• Library Design Considerations:

  • Use immune libraries from rabbits immunized with At2g40995

  • Implement synthetic libraries with optimized frameworks

  • Create libraries in both scFv and Fab formats for comparison

• Selection Strategy Optimization:

  • Employ decreasing antigen concentrations across panning rounds

  • Implement off-rate selection to identify high-affinity binders

  • Use alternating selection surfaces to reduce non-specific binders

• Screening Approaches:

  • Develop high-throughput competition ELISA for specificity assessment

  • Implement surface plasmon resonance for affinity determination

  • Use next-generation sequencing to track selection enrichment

• Antibody Engineering:

  • Convert promising scFv to full IgG format for enhanced functionality

  • Optimize framework regions for stability and expression

  • Consider affinity maturation through targeted mutagenesis

Recent advances in de novo antibody design have demonstrated success in generating antibodies with tailored properties, suggesting promising approaches for At2g40995-specific antibody development .

How might advances in computational antibody design improve At2g40995 antibody development?

Recent breakthroughs in computational antibody design offer promising approaches:

• Structure-Based Design:

  • Use protein structure prediction to model At2g40995

  • Identify optimal epitopes for antibody binding

  • Design complementary binding interfaces in antibody variable regions

• Library-Based Approaches:

  • Create rational designed antibody libraries targeting At2g40995 epitopes

  • Combine computational design with yeast display screening

  • Implement machine learning to predict binding properties

• De Novo Design Methods:

  • Apply atomic-accuracy structure prediction to design specific binders

  • Create libraries combining designed light and heavy chain sequences

  • Screen resulting libraries for binders with desired properties

Recent research demonstrates that precise, sensitive, and specific antibody design can be achieved without prior antibody information, with designed antibodies exhibiting affinity, activity, and developability comparable to commercial antibodies .

What role might At2g40995 play in climate adaptation based on current genotype-environment association research?

Emerging research on genotype-environment associations provides a framework for investigating At2g40995's potential role:

• Allele Frequency Analysis:

  • Examine At2g40995 allele distribution across moisture gradients

  • Compare sequence variation between drought-prone and moisture-rich habitats

  • Identify potentially adaptive polymorphisms

• Functional Validation Approaches:

  • Use knockout/knockdown lines to test for drought-related phenotypes

  • Measure stomatal conductance and specific leaf area under drought conditions

  • Assess flowering time responses to moisture variation

• Population Genomics Integration:

  • Analyze signatures of selection around the At2g40995 locus

  • Compare with known drought adaptation loci like WRKY38 and LSD1

  • Identify potential regulatory variants affecting expression

Recent experimental validation of genotype-environment associations in Arabidopsis has identified genes contributing to local adaptation to drought conditions, providing methodological frameworks applicable to studying At2g40995's potential adaptive roles .

How can researchers contribute to improving At2g40995 antibody resources for the scientific community?

Advancing the field requires collaborative approaches:

• Validation Data Sharing:

  • Publish comprehensive antibody validation data

  • Deposit detailed protocols in repositories like Protocols.io

  • Share negative results to prevent duplication of effort

• Resource Development:

  • Create knockout and epitope-tagged lines for antibody validation

  • Develop and share recombinant protein standards

  • Establish benchmark datasets for antibody performance evaluation

• Methodological Innovations:

  • Apply emerging antibody engineering technologies to improve specificity

  • Develop new validation approaches for plant antibodies

  • Create standardized reporting formats for antibody characterization

• Community Standards Development:

  • Establish minimum validation requirements for plant antibodies

  • Create data sharing frameworks for antibody characterization

  • Develop quality metrics for commercial and academic antibody resources