AVT6D Antibody

What is the AVT6D antibody and what is its target protein?

The AVT6D antibody is a rabbit polyclonal antibody developed against recombinant Arabidopsis thaliana AVT6D protein (UniProt Number: Q0WQJ3). This antibody is purified using Protein A/G affinity chromatography, ensuring high specificity and low background signal in experimental applications . The target AVT6D protein belongs to the amino acid transporter family in plants and is involved in cellular transport mechanisms. Understanding the function of AVT6D in plants requires reliable antibody detection methods for protein localization, expression analysis, and interaction studies.

AVT6D antibodies are typically supplied with key components that facilitate experimental validation, including:

• 200μg recombinant immunogen protein/peptide (positive control)

• 1ml pre-immune serum

• Purified rabbit polyclonal antibody

These components enable proper experimental design with appropriate controls for validating antibody specificity and interpreting experimental results with confidence.

What applications is the AVT6D antibody validated for?

The AVT6D antibody has been validated for specific research applications including ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) techniques . These methods serve different analytical purposes:

ELISA applications:

• Quantitative measurement of AVT6D protein levels in plant extracts

• High-throughput screening of multiple samples

• Detection of AVT6D protein in solution without denaturation

Western Blot applications:

• Determination of AVT6D protein molecular weight

• Analysis of AVT6D protein expression across different tissues or conditions

• Evaluation of post-translational modifications

When designing experiments, researchers should consider that the antibody's performance may vary between applications. Preliminary validation experiments are recommended to determine optimal antibody dilutions for each specific application and sample type.

What are the optimal storage and handling conditions for maintaining AVT6D antibody activity?

Proper storage and handling of the AVT6D antibody are critical for maintaining its activity and specificity. The manufacturer recommends storage at either -20°C or -80°C for long-term preservation . For optimal antibody performance and stability:

• Aliquot the antibody upon receipt to minimize freeze-thaw cycles

• Store aliquots at recommended temperatures (-20°C or -80°C)

• Transport using cold chain management (BLUE ICE shipping is recommended)

• Avoid repeated freeze-thaw cycles which can lead to aggregation and loss of activity

• When thawing, keep the antibody on ice and use immediately for experiments

• For short-term storage (1-2 weeks), antibody can be kept at 4°C

Some researchers add preservatives such as sodium azide (0.02-0.05%) to antibody solutions to prevent microbial contamination, but this should be tested to ensure it doesn't interfere with downstream applications.

How can I validate the specificity of AVT6D antibody for my experimental system?

Validating antibody specificity is crucial for meaningful experimental results. For AVT6D antibody, a comprehensive validation approach should include:

Pre-absorption testing:

• Incubate the antibody with excess purified recombinant AVT6D protein (provided as positive control)

• Use the pre-absorbed antibody in parallel with non-absorbed antibody

• Specific binding should be significantly reduced in the pre-absorbed sample

Multiple detection methods:
Cross-validate findings using different techniques. For example, if protein localization is detected by immunohistochemistry, confirm with Western blot or mass spectrometry.

Knockout/knockdown controls:
When possible, use plant material with reduced or eliminated expression of AVT6D (through CRISPR, RNAi, or T-DNA insertion lines) to confirm absence of signal.

Correlation with gene expression:
Demonstrate correspondence between protein detection and transcript levels using RT-qPCR or RNA-seq data.

Recent research on antibody specificity suggests that identifying distinct binding modes can help characterize antibody-antigen interactions. Computational models trained on experimental data can predict antibody binding profiles, which is particularly useful when working with closely related antigens .

What optimization strategies are recommended for Western blot using AVT6D antibody?

Optimizing Western blot protocols for AVT6D antibody requires systematic adjustment of multiple parameters:

Sample preparation optimization:

• Test multiple protein extraction buffers to maximize AVT6D recovery

• Compare different reducing conditions (DTT vs β-mercaptoethanol)

• Optimize protein concentration (typically 20-50 μg total protein per lane)

Blocking optimization:
Test different blocking agents to minimize background:

Antibody dilution optimization:
Perform a dilution series experiment to determine optimal antibody concentration:

• Test primary antibody at 1:500, 1:1000, 1:2000, and 1:5000 dilutions

• Test secondary antibody at manufacturer's recommended dilution range

• Select dilution that provides best signal-to-noise ratio

Incubation conditions:

• Compare overnight incubation at 4°C versus 1-2 hours at room temperature

• Test different washing buffer compositions (PBS-T versus TBS-T)

• Optimize number and duration of washing steps

Recent studies on therapeutic antibodies have shown that modification of antibody frameworks can affect specificity and binding properties . When troubleshooting, consider that even small variations in protocol can significantly impact results.

How can I use the AVT6D antibody for immunoprecipitation studies to identify protein interactions?

While the AVT6D antibody is primarily validated for ELISA and Western blot , adapting it for immunoprecipitation (IP) requires careful optimization:

IP Protocol Optimization:

• Antibody-bead coupling:

  • Test different coupling strategies (direct coupling vs. pre-bound Protein A/G)

  • Optimize antibody amount (typically 2-5 μg per reaction)

  • Consider using chemical crosslinking to prevent antibody leaching

• Sample preparation:

  • Use mild, non-denaturing lysis buffers to preserve protein interactions

  • Include protease and phosphatase inhibitors

  • Pre-clear lysates with beads alone to reduce non-specific binding

• Control experiments:

  • Include a negative control using pre-immune serum

  • Include an isotype control (non-specific rabbit IgG)

  • Validate pulled-down proteins by mass spectrometry

• Validation of interactions:

  • Confirm interactions with reciprocal IP when possible

  • Use proximity ligation assays as an orthogonal validation method

  • Consider yeast two-hybrid or split-GFP assays for further validation

Successful IP experiments with plant samples often require optimization of tissue disruption methods, as plant cell walls and secondary metabolites can interfere with antibody-antigen interactions. The research approach should be informed by methodologies used in studies of antibody binding specificity, where multiple complementary techniques provide confidence in results .

How can cross-reactivity of AVT6D antibody with homologous proteins be assessed and controlled?

Cross-reactivity assessment is essential when working with antibodies targeting members of protein families with high sequence similarity:

In silico analysis:

• Perform sequence alignment of AVT6D with homologous proteins in your species of interest

• Identify regions with high similarity that might lead to cross-reactivity

• Use epitope prediction tools to assess if the immunogen region is conserved across homologs

Experimental validation:

• Express recombinant homologous proteins as potential competitors

• Perform Western blot analysis with samples containing known homologs

• Use peptide competition assays with peptides derived from homologous proteins

Controls for experimental interpretation:

• Include samples from plants with varying expression levels of AVT6D and its homologs

• Use the pre-immune serum provided with the antibody kit as a negative control

• Design experiments to distinguish between direct binding and indirect interactions

Research on antibody specificity has demonstrated that even antibodies with high sequence similarity can have distinct binding profiles . This principle applies to antigenic targets as well—highly similar proteins may present distinctly different epitopes depending on their three-dimensional structure and post-translational modifications.

What are recommended approaches for using AVT6D antibody in plant immunohistochemistry/immunofluorescence?

Although not explicitly listed among the validated applications , researchers may adapt the AVT6D antibody for immunohistochemistry or immunofluorescence with appropriate optimization:

Tissue preparation considerations:

• Compare different fixation methods (paraformaldehyde, glutaraldehyde, or combinations)

• Test various antigen retrieval techniques (heat-induced, enzymatic, or pH-based)

• Optimize section thickness for best penetration/preservation balance

Antibody incubation optimization:

• Test longer incubation times at lower temperatures to improve specificity

• Determine optimal antibody concentration through titration experiments

• Consider signal amplification methods for low-abundance targets

Validation approaches:

• Perform parallel detection with fluorescent protein fusions when possible

• Include absorption controls with recombinant antigen

• Compare localization patterns with published transcriptomic data for tissue-specific expression

Troubleshooting common issues:

• Autofluorescence in plant tissues can be reduced using specific quenching agents

• Non-specific binding can be minimized by extensive blocking and using detergents

• Signal amplification may be necessary due to relatively low abundance of transport proteins

Studies on antibody-mediated imaging have shown that the microenvironment can significantly affect antibody binding properties , which is particularly relevant in plant tissues where cell wall components and vacuoles create distinctive physicochemical environments.

What are the most common causes of false positive/negative results when using AVT6D antibody?

Understanding potential sources of error helps researchers design robust experiments and interpret results accurately:

Common sources of false positives:

Common sources of false negatives:

Research on antibody specificity has shown that binding can be influenced by subtle changes in experimental conditions . Careful experimental design with appropriate controls is essential for confident interpretation of results.

How can I quantitatively evaluate AVT6D antibody binding characteristics?

Quantitative evaluation of antibody performance provides critical information for experimental design and interpretation:

Affinity determination methods:

• Surface Plasmon Resonance (SPR) to measure association/dissociation kinetics

• Bio-Layer Interferometry (BLI) for real-time binding analysis

• Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters

ELISA-based quantification approaches:

• Indirect ELISA with serial dilutions to determine EC50 values

• Competitive ELISA to assess relative binding affinities

• Sandwich ELISA to determine lower limits of detection

Data analysis considerations:

$K_D = \frac{k_{off}}{k_{on}}$

Where:

• K_D is the dissociation constant (measure of affinity)

• k_off is the dissociation rate constant

• k_on is the association rate constant

Understanding these parameters allows researchers to predict antibody performance under different experimental conditions and interpret results with appropriate confidence levels.

How can AVT6D antibody be used to study protein expression changes under different stress conditions?

Investigating AVT6D protein expression under various stresses can provide insights into plant stress response mechanisms:

Experimental design considerations:

• Include appropriate time course sampling to capture both early and late responses

• Use multiple biological replicates to account for plant-to-plant variation

• Include non-stressed controls for normalized comparisons

Quantification approaches:

• Western blot with densitometry for semi-quantitative analysis

• ELISA for more precise quantification

• Normalization to stable reference proteins for accurate comparisons

Data interpretation framework:

• Correlate protein level changes with transcriptomic data when available

• Consider post-translational modifications using phospho-specific or other modification-specific antibodies

• Integrate findings with physiological measurements to establish functional relevance

Recent studies have highlighted the importance of understanding protein-specific responses to stress conditions beyond transcriptional regulation . Using antibodies like AVT6D in combination with other approaches provides a more complete picture of cellular responses.

What approaches can be used to determine if post-translational modifications affect AVT6D antibody recognition?

Post-translational modifications (PTMs) can significantly affect antibody recognition and provide important biological insights:

Experimental approaches:

• Compare antibody binding to native and dephosphorylated/deglycosylated samples

• Use phosphatase or glycosidase treatments followed by Western blot analysis

• Develop modification-specific antibodies for key PTM sites

Mass spectrometry validation:

• Immunoprecipitate the protein using AVT6D antibody

• Analyze the precipitated protein by mass spectrometry to identify PTMs

• Compare PTM patterns with antibody recognition patterns

Interpreting PTM data:
The presence of PTMs might explain tissue-specific or condition-specific variations in antibody detection. Understanding these patterns can provide insights into AVT6D regulation mechanisms.

Research on therapeutic antibodies has shown that modifications can significantly affect immunogenicity and binding properties . Similar principles apply to target proteins, where PTMs can create or mask epitopes.

What are emerging technologies that might complement or enhance AVT6D antibody-based research?

Staying informed about technological advances can enhance experimental approaches:

• Single-cell proteomics:

  • Combining AVT6D antibody with single-cell technologies for spatial resolution

  • Analysis of cell-to-cell heterogeneity in AVT6D expression

• Proximity labeling approaches:

  • Antibody-guided proximity labeling to identify interacting partners

  • Spatial mapping of protein complexes containing AVT6D

• Nanobody and synthetic binding protein alternatives:

  • Development of smaller binding reagents with potentially improved tissue penetration

  • Enhanced specificity through structure-guided design

• Computational approaches:

  • Biophysics-informed models to predict and design antibody specificity

  • Integration of experimental data with computational prediction tools

As research continues to evolve, the integration of traditional antibody-based methods with emerging technologies will provide increasingly detailed insights into plant protein function and regulation.