At4g04260 Antibody

What is AT4G04260 and why is it studied in plant research?

AT4G04260 is a gene in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology and genetic research. This gene encodes a protein that has drawn research interest due to its potential role in plant developmental processes and stress responses. Antibodies against this protein are valuable tools for examining its expression patterns, localization, and functional interactions in plant tissues . Methodologically, researchers typically utilize AT4G04260 antibodies to track protein expression changes under various environmental conditions, developmental stages, or genetic backgrounds, contributing to our understanding of plant biology fundamentals.

What applications are AT4G04260 antibodies validated for?

Based on manufacturer specifications, AT4G04260 antibodies are primarily validated for Western Blot (WB) and ELISA applications, with specificity for Arabidopsis samples . For Western blotting, researchers typically use these antibodies at dilutions ranging from 1:500 to 1:2000, depending on the specific antibody and experimental conditions. ELISA applications generally employ dilutions in the 1:1000 to 1:5000 range. When designing experiments, it's essential to conduct preliminary titration experiments to determine optimal antibody concentrations for your specific sample type and detection system.

How should AT4G04260 antibodies be stored and handled to maintain reactivity?

For optimal performance, store AT4G04260 antibodies according to manufacturer specifications, typically at -20°C for long-term storage with minimal freeze-thaw cycles. When working with these antibodies:

• Aliquot upon receipt to minimize freeze-thaw cycles (typically 5-10 μL aliquots)

• Store working dilutions at 4°C for short-term use (1-2 weeks maximum)

• Use sterile techniques when handling to prevent microbial contamination

• Avoid repeated freeze-thaw cycles that can lead to antibody degradation and decreased specificity

• Centrifuge briefly before opening vials to collect liquid at the bottom of the tube

These handling practices ensure maximum antibody reactivity and experimental reproducibility when working with plant samples .

What are the typical sample preparation methods for AT4G04260 detection in Arabidopsis?

When preparing Arabidopsis samples for AT4G04260 detection, researchers should follow these methodological steps:

For Western Blot analysis:

• Harvest tissue samples (typically 100-200 mg) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder while maintaining freezing conditions

• Extract proteins using a plant-specific extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 150 mM NaCl

  • 1% Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• Centrifuge at 12,000 × g for 10 minutes at 4°C

• Collect supernatant and quantify protein concentration

• Prepare samples with reducing loading buffer and heat at 95°C for 5 minutes

For ELISA:

• Extract proteins as described above

• Dilute samples to working concentration (typically 1-10 μg/mL) in coating buffer

• Follow standard ELISA protocols with appropriate blocking agents specific for plant samples

These preparation methods maximize protein extraction while preserving epitope integrity for antibody recognition .

How can cross-reactivity be assessed when using AT4G04260 antibodies in different plant species?

Cross-reactivity assessment is critical when expanding AT4G04260 antibody applications beyond Arabidopsis. Implement this methodological approach:

• Sequence homology analysis: Compare AT4G04260 protein sequences across target species using alignment tools (BLAST, Clustal Omega). Aim for >70% homology in antibody epitope regions.

• Validation experiments:

  • Perform Western blots using recombinant AT4G04260 protein as a positive control

  • Include samples from both Arabidopsis and target species

  • Include knockout/knockdown samples as negative controls where available

  • Analyze molecular weight differences and band patterns

• Competitive blocking:

  • Pre-incubate antibody with recombinant AT4G04260 protein

  • Apply to parallel samples to confirm specificity

• Immunoprecipitation validation:

  • Perform IP followed by mass spectrometry to confirm target identity in new species

Cross-reactivity data should be presented as shown in this example table:

This systematic approach ensures reliable interpretation of cross-reactivity results and avoids false positives or negatives when working with AT4G04260 antibodies across species .

What are the best practices for optimizing immunolocalization using AT4G04260 antibodies?

While the current AT4G04260 antibodies are primarily validated for WB and ELISA, researchers may adapt them for immunolocalization with careful optimization:

• Fixation optimization:

  • Test multiple fixatives: 4% paraformaldehyde, Carnoy's solution, and glutaraldehyde-based fixatives

  • Vary fixation duration (30 minutes to overnight at 4°C)

  • Evaluate epitope preservation through control experiments

• Antigen retrieval methods:

  • Heat-mediated retrieval: Test citrate buffer (pH 6.0) and Tris-EDTA (pH 9.0)

  • Enzymatic retrieval: Try proteinase K (1-5 μg/mL) for 5-15 minutes

  • Document retrieval efficiency for each condition

• Blocking optimization:

  • Test BSA (3-5%), normal serum (5-10%), and commercial blocking reagents

  • Include plant-specific blocking steps to reduce background (pre-incubation with extract from knockout plants)

• Antibody titration:

  • Create a dilution series (1:50 to 1:1000)

  • Include appropriate positive and negative controls

  • Document signal-to-noise ratio for each dilution

• Detection system comparison:

  • Fluorescent secondary antibodies (Alexa Fluor series)

  • Enzymatic detection (HRP/DAB or AP)

  • Amplification systems (tyramide signal amplification)

Document optimization using a systematic approach that records all parameters and their effect on signal intensity, background, and target localization specificity .

How can quantitative analysis be performed with AT4G04260 antibodies?

For quantitative analysis of AT4G04260 protein levels, researchers should follow these methodological guidelines:

• Western blot quantification:

  • Use internal loading controls (housekeeping proteins like actin or tubulin)

  • Prepare standard curves with recombinant protein if available

  • Employ digital image analysis software (ImageJ/FIJI) with appropriate background correction

  • Calculate relative or absolute protein quantities using the following formula:
    $\text{Relative quantity} = \frac{\text{Target protein band intensity}}{\text{Loading control band intensity}}$

• Quantitative ELISA:

  • Develop a standard curve using recombinant AT4G04260 protein

  • Ensure linear range detection (typically 0.1-1000 ng/mL depending on antibody affinity)

  • Calculate protein concentration using the standard curve equation:
    $\text{Concentration} = \text{f}(\text{Absorbance})$

  • Include technical and biological replicates (minimum n=3)

• Data normalization approaches:

  • Total protein normalization (recommended for plant samples with variable housekeeping protein expression)

  • Multiple reference gene approach for more accurate normalization

  • Consider tissue-specific reference proteins for specialized plant tissues

• Statistical analysis:

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Calculate confidence intervals for all measurements

  • Report both biological and technical variation

These methodological approaches ensure accurate and reproducible quantification of AT4G04260 protein levels across experimental conditions .

How can AT4G04260 antibodies be used to investigate protein-protein interactions?

To investigate AT4G04260 protein-protein interactions, researchers can utilize these antibodies in several advanced applications:

• Co-immunoprecipitation (Co-IP):

  • Prepare plant lysates under native conditions using mild detergents (0.5% NP-40 or 0.1% Triton X-100)

  • Incubate lysates with AT4G04260 antibody coupled to protein A/G beads

  • Analyze precipitated complexes by mass spectrometry or Western blot

  • Validate interactions with reverse Co-IP using antibodies against putative interacting partners

• Proximity ligation assay (PLA):

  • Combine AT4G04260 antibody with antibodies against suspected interaction partners

  • Utilize species-specific PLA probes with oligonucleotide tags

  • Amplify and detect signals only when proteins are in close proximity (<40 nm)

  • Quantify interaction events through fluorescent dot counting

• Bimolecular Fluorescence Complementation (BiFC) validation:

  • Use antibody data to guide selection of protein pairs for BiFC analysis

  • Design complementary fusion constructs for AT4G04260 and potential partners

  • Validate interaction sites identified by antibody-based methods

• Analytical techniques:

  • Blue Native PAGE followed by antibody detection to preserve native complexes

  • Size-exclusion chromatography with fraction analysis by immunoblotting

  • Chemical crosslinking followed by immunoprecipitation (CLIP)

These approaches provide complementary data on AT4G04260 protein interactions, with each method offering different advantages in specificity, sensitivity, and in vivo relevance .

What are the common sources of false positives/negatives when using AT4G04260 antibodies?

When working with AT4G04260 antibodies, researchers should be aware of these potential sources of experimental artifacts:

Sources of false positives:

• Cross-reactivity with homologous proteins in Arabidopsis

• Non-specific binding to abundant plant proteins (particularly problematic in ELISA)

• Secondary antibody cross-reactivity with endogenous plant immunoglobulins

• Insufficient blocking, particularly with plant samples containing high polysaccharide content

• Post-translational modifications altering epitope recognition

Sources of false negatives:

• Epitope masking due to protein-protein interactions or conformational changes

• Protein degradation during sample preparation

• Insufficient extraction from plant cell walls or subcellular compartments

• Fixation-induced epitope destruction in immunohistochemistry

• Antibody degradation due to improper storage or handling

Validation approaches:

• Include knockout/knockdown samples as negative controls

• Perform peptide competition assays to confirm specificity

• Validate results with multiple antibodies targeting different epitopes

• Correlate protein detection with transcript levels (though not always concordant)

• Use recombinant protein as a positive control

This comprehensive validation strategy ensures reliable interpretation of AT4G04260 antibody results and prevents experimental artifacts .

How can immunoblotting conditions be optimized for AT4G04260 detection in plant samples?

Optimizing immunoblotting conditions for AT4G04260 detection requires systematic adjustment of multiple parameters:

• Sample preparation refinement:

  • Test multiple extraction buffers with varying detergent concentrations

  • Evaluate different reducing agent concentrations

  • Compare fresh vs. frozen sample processing

  • Document protein recovery with each method

• Gel and transfer optimization:

  • Test gradient gels (4-12%, 4-20%) for optimal separation

  • Compare wet and semi-dry transfer methods

  • Evaluate transfer buffers with varying methanol concentrations (10-20%)

  • Optimize transfer time and voltage for complete protein transfer

• Blocking strategy:

  • Compare milk vs. BSA vs. plant-specific blocking reagents

  • Test blocking duration (1 hour to overnight)

  • Evaluate blocking temperature (room temperature vs. 4°C)

• Antibody incubation parameters:

  • Create an antibody dilution matrix (1:500 to 1:5000)

  • Test incubation duration (1 hour to overnight)

  • Compare incubation temperatures (4°C, room temperature)

  • Evaluate diluent composition (with/without detergents, protein carriers)

• Detection system selection:

  • Compare chemiluminescence, fluorescence, and chromogenic detection

  • Test signal enhancement systems for low abundance targets

  • Evaluate digital imaging vs. film-based detection

Researchers should document all optimization steps systematically, creating a reference protocol for reproducible AT4G04260 detection in their specific experimental system .

What controls and validation methods are essential for publications using AT4G04260 antibodies?

For publication-quality research using AT4G04260 antibodies, these controls and validation methods are essential:

• Antibody validation controls:

  • Knockout/knockdown samples as negative controls

  • Overexpression systems as positive controls

  • Peptide competition/blocking experiments

  • Multiple antibodies targeting different epitopes when available

  • Recombinant protein standards for size verification

• Experimental design controls:

  • Biological replicates (minimum n=3) with statistical analysis

  • Technical replicates to assess method variability

  • Loading/extraction controls (total protein stains, housekeeping proteins)

  • Sample processing controls (freshly prepared vs. stored samples)

• Orthogonal validation methods:

  • Correlation with transcript levels (qPCR, RNA-seq)

  • Mass spectrometry validation of detected bands

  • Alternative detection methods (activity assays if applicable)

  • Genetic complementation studies

• Documentation requirements:

  • Complete antibody information (supplier, catalog number, lot, dilution)

  • Detailed methodological parameters (incubation times, temperatures, buffers)

  • Full blot/gel images with molecular weight markers

  • Quantification methods and statistical analysis details

• Reproducibility considerations:

  • Independent biological replicates

  • Verification across different growth conditions or developmental stages

  • Batch effects documentation and control

This comprehensive validation approach ensures publication-quality data and facilitates reproducibility by other research groups .

How can AT4G04260 antibodies be adapted for high-throughput plant phenotyping?

Adapting AT4G04260 antibodies for high-throughput plant phenotyping requires methodological innovations:

• Microplate-based ELISA adaptation:

  • Develop simplified protein extraction protocols compatible with 96/384-well formats

  • Optimize minimal sample requirements (50-100 μg tissue)

  • Implement robotic liquid handling for consistent processing

  • Develop standardized calibration curves for quantitative analysis

• Tissue microarray applications:

  • Create plant tissue microarrays with multiple samples embedded in paraffin

  • Adapt immunohistochemistry protocols for simultaneous processing

  • Develop automated image acquisition and quantification workflows

  • Establish normalization standards across arrays

• Multiplex detection systems:

  • Combine AT4G04260 antibody with antibodies against other markers

  • Utilize differentially labeled secondary antibodies

  • Develop spectral unmixing protocols for overlapping fluorophores

  • Create reference standards for quantification

• Data integration approaches:

  • Correlate protein expression with phenotypic parameters

  • Develop machine learning algorithms for pattern recognition

  • Integrate immunological data with other -omics datasets

  • Create standardized data repositories for cross-study comparisons

This systematic approach enables scaling from individual experiments to population-level studies of AT4G04260 expression patterns and their correlation with plant phenotypes .

What emerging technologies may enhance AT4G04260 protein research beyond traditional antibody applications?

Several emerging technologies show promise for enhancing AT4G04260 protein research:

• CRISPR epitope tagging strategies:

  • Endogenous tagging of AT4G04260 with epitope tags (FLAG, HA, GFP)

  • Generation of knock-in lines with minimal disruption to native expression

  • Utilization of well-validated tag-specific antibodies

  • Advantages: circumvents antibody specificity issues, enables live imaging

• Nanobody development:

  • Generation of camelid single-domain antibodies against AT4G04260

  • Engineering for intracellular expression and tagging

  • Applications in live-cell imaging and protein dynamics

  • Benefits: smaller size, enhanced tissue penetration, intracellular stability

• Proximity-dependent labeling:

  • TurboID or APEX2 fusion to AT4G04260

  • Mapping protein neighborhoods in living cells

  • Mass spectrometry identification of proximal proteins

  • Advantage: captures transient and weak interactions missed by Co-IP

• Single-molecule detection methods:

  • Super-resolution microscopy with antibody-based detection

  • Quantitative single-molecule localization microscopy

  • Correlation with functional parameters at subcellular resolution

  • Benefit: reveals spatial organization beyond diffraction limit

• Advanced mass spectrometry:

  • Targeted proteomics approaches (PRM, MRM)

  • Label-free quantification for improved sensitivity

  • Post-translational modification mapping

  • Advantage: orthogonal validation independent of antibody specificity

These emerging approaches complement traditional antibody applications while addressing limitations in specificity, sensitivity, and throughput .

What are the most significant challenges and future perspectives in AT4G04260 antibody research?

The current landscape of AT4G04260 antibody research presents several significant challenges and opportunities:

Current challenges:

• Limited validation across diverse plant tissues and developmental stages

• Incomplete characterization of post-translational modifications and their effect on antibody recognition

• Cross-reactivity concerns with homologous proteins in non-model plant species

• Variability between antibody lots affecting experimental reproducibility

• Limited availability of antibodies targeting different epitopes for comprehensive validation

Future research directions:

• Development of monoclonal antibodies with enhanced specificity and lot-to-lot consistency

• Comprehensive epitope mapping to better understand antibody-target interactions

• Validation across broader phylogenetic diversity to enable comparative plant biology

• Integration with emerging -omics technologies for systems-level analysis

• Application to agricultural research beyond model organisms

Technological innovations:

• Recombinant antibody technologies for improved reproducibility

• Synthetic biology approaches for novel detection reagents

• Microfluidic platforms for enhanced sensitivity and reduced sample requirements

• Computational tools for antibody design and epitope prediction

• Open science initiatives for antibody validation and data sharing