At1g23390 Antibody

What is the At1g23390 protein and what organism systems can it be studied in?

At1g23390 encodes an F-box/kelch-repeat protein initially identified in Arabidopsis thaliana (hence the "At" prefix) but also found in other plant species such as Solanum lycopersicum (tomato). The protein contains characteristic F-box domains that typically function in protein-protein interactions and ubiquitin-mediated degradation pathways . For experimental systems, researchers can work with both Arabidopsis and tomato models, with the protein sequence being conserved enough to allow cross-species antibody recognition in many cases.

What methods are typically used to generate antibodies against plant proteins like At1g23390?

Antibodies against plant proteins such as At1g23390 are commonly generated through:

• Recombinant protein immunization: The target protein is expressed in bacterial systems, purified, and used as an immunogen in mice or rabbits

• Synthetic peptide immunization: Short, unique peptide sequences from the At1g23390 protein are synthesized and conjugated to carrier proteins before immunization

• DNA immunization: Expression vectors containing the At1g23390 gene are used to immunize animals

For plant-specific proteins like F-box/kelch-repeat proteins, researchers often choose epitopes from unique regions that don't share homology with host animal proteins to reduce cross-reactivity .

What experimental applications are suitable for At1g23390 antibodies?

At1g23390 antibodies are versatile tools for multiple experimental applications:

These applications should be validated with appropriate positive and negative controls for each experimental system .

How can chromatin configuration affect experimental outcomes when studying F-box proteins like At1g23390?

Chromatin configuration significantly impacts experiments involving F-box proteins like At1g23390, particularly when studying their role in transcriptional regulation. Research has demonstrated that DNA organization, together with proteins forming chromatin, influences protein accessibility and function . When designing ChIP experiments with At1g23390 antibodies, researchers should consider:

• Chromatin loop formation mediated by cohesin complexes may regulate accessibility to F-box protein binding sites

• CTCF-binding elements (CBEs) that define loop domains may impact F-box protein distribution

• Different crosslinking methods may preferentially capture specific chromatin configurations

Researchers should incorporate sonication optimization steps that account for variations in chromatin compaction across different plant tissues and developmental stages .

What methodological approaches can resolve specificity issues with At1g23390 antibodies?

Resolving specificity issues with At1g23390 antibodies requires systematic approach:

• Epitope mapping validation: Perform competitive binding assays with the immunizing peptide to confirm epitope specificity

• Knockout validation: Test antibody in At1g23390 knockout/knockdown tissues to confirm absence of signal

• Mass spectrometry validation: Analyze immunoprecipitation products by MS to confirm target identity

• Isotype-matched control experiments: Use matched IgM (for monoclonal) or pre-immune serum (for polyclonal) controls

• Cross-adsorption: Pre-incubate antibody with related proteins to remove cross-reactive antibodies

• Multiple antibody approach: Use antibodies targeting different epitopes of At1g23390 to confirm results

These approaches can significantly enhance confidence in experimental results and should be documented in publications .

How can transcriptome analysis complement At1g23390 antibody-based studies?

Integrating transcriptome analysis with At1g23390 antibody studies provides a powerful multi-omics approach:

• Correlation analysis: Compare protein levels (detected by At1g23390 antibody) with mRNA expression levels to identify post-transcriptional regulation

• Differential expression after perturbation: Analyze transcriptome changes following At1g23390 knockdown/overexpression to identify genes regulated by this F-box protein

• Protein-RNA interaction studies: Combine RNA immunoprecipitation with At1g23390 antibodies followed by RNA-seq to identify RNA targets

• Time-course experiments: Following the approach in search result , perform time-course experiments (0, 4, 12, 24h) after treatment to capture dynamic responses

A study using similar approaches identified 1,385, 734, and 6,109 differentially expressed genes at different time points following experimental treatment, demonstrating the power of combining antibody studies with transcriptomics .

What are the most common causes of inconsistent results when using At1g23390 antibodies?

Inconsistent results with At1g23390 antibodies typically stem from several factors:

• Sample preparation variability: Different extraction methods can affect protein conformation and epitope accessibility

• Post-translational modifications: F-box proteins undergo regulatory modifications including phosphorylation and ubiquitination that may mask epitopes

• Antibody lot-to-lot variation: Monoclonal antibodies show less variation than polyclonal, but both can exhibit batch effects

• Buffer compatibility issues: Plant samples contain phenolics and other compounds that can interfere with antibody binding

• Fixation artifacts: Overfixation can destroy epitopes while underfixation risks protein loss

Methodological solution: Implement a rigorous validation protocol for each new antibody lot using positive controls (recombinant At1g23390) and negative controls (knockout tissue extracts) before proceeding with experiments .

How should plant tissue samples be prepared to optimize At1g23390 antibody detection?

Optimal plant tissue preparation for At1g23390 detection requires specific considerations:

• Harvest timing: Collect samples at consistent times to control for circadian regulation of F-box proteins

• Flash freezing: Immediately freeze tissues in liquid nitrogen to prevent protein degradation

• Extraction buffer optimization:

  • Include protease inhibitors (PMSF, leupeptin, aprotinin)

  • Add phosphatase inhibitors when studying phosphorylation states

  • Include reducing agents (DTT or β-mercaptoethanol) to maintain protein conformation

  • Add PVPP (polyvinylpolypyrrolidone) to remove phenolic compounds

• Mechanical disruption: Use bead beaters or tissue homogenizers while keeping samples cold

• Subcellular fractionation: Consider separate nuclear, cytoplasmic, and membrane fractions as F-box proteins may shuttle between compartments

This approach has been successfully used in studies examining plant proteins with similar characteristics to At1g23390 .

What controls are essential when analyzing protein-protein interactions involving At1g23390?

Essential controls for protein-protein interaction studies include:

• Input control: Analyze a portion of the starting material to confirm target presence

• IP antibody isotype control: Use matched isotype antibody to identify non-specific binding

• Bead-only control: Include sample processed without antibody to detect bead-binding proteins

• Reciprocal IP: Confirm interactions by IP with antibodies against putative interaction partners

• Competition control: Addition of excess immunizing peptide should abolish specific signals

• Denaturing control: Compare native vs. denaturing conditions to distinguish direct interactions

For interactions involving F-box proteins like At1g23390, additional controls should address transient interactions that may occur only under specific conditions (e.g., during ubiquitination) .

How should quantitative At1g23390 protein data be normalized across different experimental conditions?

Proper normalization of At1g23390 protein quantification requires:

• Loading control selection: Use stable reference proteins such as β-actin or GAPDH for western blot normalization

• Normalization factor validation: Verify stability of reference genes across experimental conditions using approaches similar to those in transcriptome studies

• Total protein normalization: Consider stain-free technology or Ponceau staining as alternatives to single reference proteins

• Statistical handling of technical replicates: Average technical replicates before analyzing biological replicates

• Normalization algorithm selection:

Researchers should validate their normalization approach using spike-in controls when possible .

What bioinformatic approaches help identify potential interactors and substrates of At1g23390?

Advanced bioinformatic approaches for studying At1g23390 interactions include:

• Protein-protein interaction network analysis: Integrate experimental data with existing PPI databases

• Protein domain prediction: Identify proteins with domains known to interact with F-box/kelch-repeat domains

• Co-expression network analysis: Identify genes whose expression patterns correlate with At1g23390

• Evolutionary conservation analysis: Compare potential interactions across species

• Structural modeling: Predict interaction interfaces based on protein structure

These approaches can be integrated with quantitative proteomics data from immunoprecipitation studies to prioritize candidates for experimental validation. Similar approaches have successfully identified key interaction networks in other systems .

How can contradictory results between antibody-based detection and transcriptome data for At1g23390 be reconciled?

Reconciling contradictory protein and transcript data requires systematic analysis:

• Temporal dynamics: Protein changes often lag behind transcript changes; time-course experiments can identify offset patterns

• Post-transcriptional regulation: Investigate microRNA targeting, RNA stability, and translational efficiency

• Post-translational regulation: Assess protein stability, degradation rates, and modifications

• Technical validation: Confirm results using alternative methods:

  • Verify antibody specificity with recombinant proteins

  • Confirm transcript quantification with qRT-PCR

• Biological context: Consider tissue-specific or cell-type-specific differences that may be masked in whole-tissue analyses

Quantitative RT-PCR can verify RNA-Seq results, as demonstrated in search result , which showed strong correlation between these methods for differentially expressed genes .

How might new antibody technologies enhance At1g23390 research?

Emerging antibody technologies with potential applications for At1g23390 research include:

• nanobodies/single-domain antibodies: Smaller size allows access to previously inaccessible epitopes

• Proximity labeling antibodies: Conjugated with enzymes like BioID or APEX to identify proteins in proximity to At1g23390

• Antibody-based biosensors: For real-time monitoring of At1g23390 dynamics in living cells

• Intrabodies: Engineered to function within cells for visualizing endogenous At1g23390

• Multiplexed antibody arrays: For studying At1g23390 in the context of broader signaling networks

These approaches could significantly advance our understanding of F-box protein dynamics in plant systems .

What methodological advances could improve chromatin studies involving At1g23390?

Recent advances in chromatin research methodologies applicable to At1g23390 studies include:

• CUT&RUN/CUT&Tag: More sensitive alternatives to traditional ChIP for mapping protein-DNA interactions

• Hi-C and derivatives: For studying three-dimensional chromatin organization relevant to F-box protein function

• Single-cell approaches: To examine cell-type-specific roles of At1g23390

• CRISPR-based techniques: For targeted modification of At1g23390 binding sites in the genome

• Live-cell chromatin imaging: For real-time visualization of At1g23390 interactions with chromatin