Target protein: At4g26020 (UniProt: Q1PE49), also termed At-4/1 or Tomato spotted wilt virus movement protein-interacting protein 4/1 .
Antibody type: Rabbit polyclonal antibody validated for ELISA and Western Blot (WB) applications .
Gene details:
The At4g26020 antibody has been instrumental in elucidating the role of At-4/1 in viral pathogenesis:
Colocalization with viral movement proteins: Confocal microscopy using GFP-tagged At-4/1 demonstrated its colocalization with Poa semilatent virus TGBp3 movement protein in Nicotiana benthamiana cells, suggesting its involvement in endoplasmic reticulum-derived membrane structures critical for viral transport .
Immunogold labeling: Electron microscopy with anti-At-4/1 antibodies revealed the protein’s association with vesicular membranes near plasmodesmata, highlighting its role in intercellular communication .
Specificity: The antibody shows minimal cross-reactivity in preimmune serum controls, with a signal-to-noise ratio >100:1 in membrane structures .
Batch consistency: Commercial batches (e.g., MyBioSource MBS7191047) are validated for lot-to-lot reproducibility in WB and ELISA .
Antibody validation: Studies emphasize the importance of rigorous validation, as highlighted by initiatives like YCharOS, which advocate for knockout controls and application-specific testing to confirm antibody specificity .
Structural insights: While not directly studied for At4g26020, advancements in antibody databases (e.g., AbDb) underscore the need for standardized annotation of antibody-antigen complexes, particularly for plant proteins .
At4g26020 encodes a protein with regulatory functions in plant stress responses similar to ASK1 (Apoptosis Signal-regulating Kinase 1) in mammalian systems. Antibodies against this protein are valuable for investigating stress signaling pathways in plants. Like the human ASK1 antibody, which plays a crucial role in cellular stress responses and apoptosis by activating downstream MAP kinase pathways , antibodies targeting At4g26020 enable researchers to study plant stress response mechanisms through various detection techniques. These antibodies facilitate the investigation of protein expression, localization, and interaction partners under different environmental stress conditions.
At4g26020 antibodies can be utilized with multiple detection methodologies similar to those employed for other research antibodies. These include:
Western blotting (WB): For detecting and quantifying protein expression levels
Immunoprecipitation (IP): For studying protein-protein interactions
Immunofluorescence (IF): For examining subcellular localization
Immunohistochemistry with paraffin-embedded sections (IHC-P): For tissue-specific expression analysis
Enzyme-linked immunosorbent assay (ELISA): For quantitative protein detection
The selection of appropriate detection method depends on your experimental objectives and available plant material. For instance, western blotting is ideal for protein expression analysis across different stress treatments, while immunofluorescence is preferable for studying subcellular localization changes in response to environmental stimuli.
Validating antibody specificity is critical for reliable experimental results. Recommended validation protocols include:
Western blot analysis using wild-type plants and At4g26020 knockout/knockdown mutants
Peptide competition assays to confirm epitope specificity
Immunoprecipitation followed by mass spectrometry analysis
Cross-reactivity testing against closely related proteins
Evaluation across multiple detection methods to ensure consistent results
Proper validation is particularly important because plant proteins often exist in multigene families with high sequence homology, which can lead to cross-reactivity. When validating, it's advisable to use multiple tissue types and developmental stages to verify antibody performance across different experimental conditions.
For optimal stability and performance, store At4g26020 antibodies following these guidelines:
Storage Parameter | Primary Antibody | Conjugated Antibody | Working Solution |
---|---|---|---|
Temperature | -20°C | 4°C | 4°C |
Buffer | PBS with 50% glycerol | PBS with 0.09% sodium azide | PBS with 0.1% BSA |
Aliquoting | Recommended (10-50 μl) | Recommended | Fresh preparation |
Freeze-thaw cycles | Minimize (<5) | Avoid | N/A |
Shelf life | 12-24 months | 6-12 months | 1-2 weeks |
Proper storage prevents antibody degradation and maintains consistent performance across experiments . Always centrifuge antibody vials briefly before opening to collect solution at the bottom of the vial.
For advanced protein interaction studies, researchers can employ At4g26020 antibodies in several sophisticated approaches:
Co-immunoprecipitation coupled with tandem mass spectrometry (Co-IP-MS/MS) to identify novel interaction partners under different stress conditions
Proximity labeling techniques such as BioID or APEX using At4g26020 antibodies for validation
Chromatin immunoprecipitation (ChIP) analysis if At4g26020 functions in transcriptional regulation complexes
Förster resonance energy transfer (FRET) microscopy using fluorophore-conjugated antibodies to validate direct protein interactions in vivo
These approaches can reveal how At4g26020 interactions change during plant development or stress responses. For example, using At4g26020 antibodies in IP followed by mass spectrometry can identify differentially associated proteins under drought versus salt stress conditions, providing insights into stress-specific signaling networks.
When investigating post-translational modifications (PTMs) of At4g26020, consider the following approaches:
Phospho-specific antibodies: For detecting specific phosphorylation sites predicted by bioinformatic analysis or identified through phosphoproteomic studies
PTM-enrichment strategies: Including phosphopeptide enrichment (TiO₂ or IMAC) prior to immunoprecipitation
2D gel electrophoresis: To separate protein isoforms based on charge differences resulting from PTMs
Phos-tag gel electrophoresis: For separating phosphorylated forms from non-phosphorylated protein
Studies of ASK1 have shown that phosphorylation significantly impacts its activity in stress signaling , suggesting similar regulation may occur for At4g26020. When analyzing samples for PTMs, carefully control protein extraction conditions to preserve labile modifications, and consider using phosphatase inhibitors during sample preparation.
Epitope masking is a common challenge when using antibodies in fixed plant tissues. Advanced solutions include:
Optimized antigen retrieval protocols specific for plant tissues:
Heat-induced epitope retrieval (HIER): Test different buffer systems (citrate buffer pH 6.0, Tris-EDTA pH 9.0) and heating methods
Enzymatic epitope retrieval: Evaluate protease K, trypsin, or plant cell wall-degrading enzymes at varying concentrations
Different fixation strategies:
Paraformaldehyde concentration optimization (1-4%)
Alternative fixatives (Carnoy's solution, methanol-acetone mixtures)
Duration of fixation optimization to minimize overfixation
Tissue clearing techniques:
The thick plant cell wall and cuticle present unique challenges for antibody penetration. Document all optimization steps meticulously, as different plant tissues may require distinct protocols for optimal epitope accessibility.
Engineering bispecific antibodies (bsAbs) targeting At4g26020 and its interaction partners requires careful design considerations:
Molecular geometry selection:
Symmetric vs. asymmetric configurations based on target proximity
IgG-like scaffolds vs. fragment-based designs depending on tissue penetration requirements
Chain pairing strategies:
"Knobs-into-holes" technology for asymmetric designs
Common light chain approach for symmetric designs
Single-chain Fv (scFv) fusions for reduced mispairing risk
Developability evaluation:
Non-specific binding is a frequent challenge with plant protein antibodies. Advanced troubleshooting approaches include:
Optimized blocking strategies:
Test different blocking agents (BSA, casein, plant-derived blockers)
Extend blocking time (overnight at 4°C)
Use commercial plant-specific blocking reagents
Pre-adsorption techniques:
Pre-incubate antibody with plant extract from knockout/knockdown lines
Use acetone powder from related plant species for pre-clearing
Buffer optimization:
Systematic testing of these parameters can significantly improve signal-to-noise ratio. Document optimal conditions thoroughly, as they may vary between plant tissues, developmental stages, and experimental conditions.
When At4g26020 antibodies perform inconsistently across different plant tissues, consider these advanced approaches:
Tissue-specific protein extraction protocols:
Modify extraction buffers based on tissue composition (higher detergent for waxy tissues)
Use specialized extraction methods for recalcitrant tissues (phenol extraction for high-proteolytic tissues)
Fixation and permeabilization optimization:
Adjust fixation time and concentration for different tissue densities
Test vacuum infiltration of fixatives for thicker tissues
Employ targeted permeabilization with cellulases/pectinases for specific cell types
Signal amplification techniques:
Different plant tissues contain varying levels of interfering compounds (phenolics, alkaloids, etc.) that can affect antibody performance. Maintain a detailed record of tissue-specific optimization parameters to ensure reproducible results across experiments.
Minimizing the impact of batch-to-batch variability requires systematic approaches:
Comprehensive characterization of new antibody batches:
Quantitative comparison of binding affinity using SPR or BLI
Epitope mapping confirmation
Side-by-side testing with previous batches across multiple applications
Internal reference standards establishment:
Create standard curves with recombinant At4g26020 protein
Maintain positive control samples from successful experiments
Implement loading normalization using constitutively expressed proteins
Statistical approaches:
Batch-to-batch variability is particularly important to address in longitudinal studies spanning multiple antibody lots. Establishing a comprehensive antibody validation protocol can help mitigate these variations and ensure consistent experimental results.
Post-translational modifications can significantly alter the chromatographic profile of At4g26020, creating acidic and basic species that may affect antibody recognition:
Common modifications creating acidic species:
Deamidation (Asn → Asp/isoAsp)
Sialylation of glycans
Oxidation of methionine residues
Formation of trisulfide bonds
C-terminal lysine clipping
Modifications creating basic species:
These modifications can affect antibody binding efficiency by altering epitope structure or accessibility. When analyzing At4g26020 using chromatographic methods, consider employing charge-based separation techniques like ion-exchange chromatography to characterize different protein species before immunodetection.
Acidic and basic species variants of At4g26020 can significantly impact antibody recognition in various ways:
Epitope accessibility effects:
Charge alterations may cause conformational changes affecting antibody binding
PTMs within or adjacent to epitopes can directly block antibody recognition
Changes in protein stability may expose or hide epitopes
Functional implications:
Deamidation in CDR regions can reduce antigen binding affinity by 14-60%
Modified disulfide bonds alter protein conformation and thermal stability
High mannose content glycoforms may appear in acidic fractions despite no charge difference
Detection strategy adjustments:
When studying stress-responsive proteins like At4g26020, be particularly aware that environmental conditions can induce PTMs. Consider developing validation strategies that account for potential stress-induced modifications to ensure consistent antibody performance across experimental conditions.
The development and application of At4g26020 antibodies continue to evolve with several promising directions:
Advanced imaging applications:
Super-resolution microscopy for nanoscale protein localization
Live-cell imaging using cell-permeable antibody fragments
Correlative light and electron microscopy for ultrastructural studies
Single-cell applications:
Antibody-based single-cell proteomics
Mass cytometry (CyTOF) adaptation for plant cell populations
In situ proximity ligation assays for protein interactions at cellular resolution
Integration with emerging technologies: