At4g17200 Antibody

What is the At4g17200 gene and its protein function in Arabidopsis thaliana?

The At4g17200 gene in Arabidopsis thaliana encodes a protein of currently unknown function, though its nomenclature suggests potential roles in plant-specific metabolic or regulatory pathways. Current research efforts are still investigating its precise biological function. When working with this antibody, researchers should note that the limited functional annotation presents both challenges and opportunities in experimental design and interpretation. Preliminary characterization suggests this protein may be involved in developmental processes or stress responses based on expression patterns, though definitive functional studies are needed to confirm these hypotheses.

How is the At4g17200 Antibody produced and what are its key properties?

The At4g17200 antibody is typically produced in mammalian hosts such as rabbits or mice, with epitopes designed against specific peptide sequences of the target protein. The production process involves immunizing host animals with purified peptide corresponding to unique sequences of the At4g17200 protein, followed by isolation and purification of the resulting antibodies. The antibody is available in liquid form, formulated in a buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative. This formulation ensures stability during storage and handling while maintaining binding activity.

What is the species reactivity profile of the At4g17200 Antibody?

The At4g17200 antibody has been primarily validated for Arabidopsis thaliana. Cross-reactivity with other plant species remains largely undocumented, representing a gap in current research. When designing experiments with non-Arabidopsis species, it's essential to include appropriate controls to validate specificity, such as:

• Western blot comparison between Arabidopsis and target species lysates

• Peptide competition assays to confirm binding specificity

• Cross-validation with other detection methods when possible

The degree of protein sequence conservation between species will likely determine cross-reactivity potential, with closely related Brassicaceae family members having higher probability of successful detection.

How can I optimize Western blot protocols for At4g17200 Antibody?

Optimizing Western blot conditions for the At4g17200 antibody requires systematic adjustment of several parameters to maximize signal-to-noise ratio:

Table 1: Recommended Western Blot Parameters for At4g17200 Antibody

For troubleshooting non-specific bands, consider:

• Pre-adsorbing antibody with knockout/knockdown Arabidopsis lysate

• Adding 0.1% SDS to antibody dilution buffer to reduce hydrophobic interactions

• Using gradient gels to better resolve proteins of similar molecular weight

What approaches can validate At4g17200 Antibody specificity in experimental systems?

Rigorous validation of antibody specificity is essential for reliable experimental results. For the At4g17200 antibody, implement a multi-faceted validation strategy:

• Genetic validation: Compare protein detection between wild-type and At4g17200 knockout/knockdown Arabidopsis lines. The absence or reduction of signal in mutant lines strongly supports specificity.

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to samples. Specific signals should be significantly reduced or eliminated.

• Recombinant protein controls: Express and purify the At4g17200 protein (or epitope-containing fragment) as a positive control to verify antibody recognition.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of proteins recognized by the antibody, particularly useful for identifying potential cross-reactivity.

• Correlation with transcript levels: Compare protein expression patterns with mRNA levels across tissues or conditions, with concordant patterns supporting specificity.

Documentation of these validation steps in publications enhances data credibility and reproducibility, particularly given the limited published literature on At4g17200 antibody applications.

How can I implement immunoprecipitation protocols with At4g17200 Antibody?

While immunoprecipitation using the At4g17200 antibody has not been extensively documented in literature, researchers can optimize protocols based on established plant protein immunoprecipitation methods:

Protocol outline:

• Tissue preparation:

  • Harvest 1-2g fresh Arabidopsis tissue

  • Flash-freeze in liquid nitrogen and grind to fine powder

  • Add 3ml extraction buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, 1mM EDTA, protease inhibitors)

  • Clarify lysate by centrifugation (14,000 × g, 15 minutes, 4°C)

• Pre-clearing:

  • Incubate lysate with 50μl protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation (1,000 × g, 5 minutes)

• Antibody binding:

  • Divide lysate into experimental and control samples

  • Add 3-5μg At4g17200 antibody to experimental sample

  • Add equivalent amount of non-specific IgG to control sample

  • Incubate overnight at 4°C with gentle rotation

• Immunoprecipitation:

  • Add 50μl pre-washed protein A/G beads to each sample

  • Incubate 3 hours at 4°C with rotation

  • Collect beads by centrifugation (1,000 × g, 2 minutes)

• Washing and elution:

  • Wash beads 4× with wash buffer (same as extraction buffer with 0.1% NP-40)

  • Elute bound proteins with 50μl 2× SDS sample buffer (95°C, 5 minutes)

  • Analyze by SDS-PAGE and Western blotting

Successful immunoprecipitation can identify protein interaction partners of the At4g17200 protein, potentially providing functional insights into this understudied protein.

What strategies should I employ for immunofluorescence with At4g17200 Antibody in plant tissues?

Immunofluorescence microscopy with plant tissues presents unique challenges due to cell wall barriers and autofluorescence. The following protocol adaptations optimize At4g17200 antibody performance:

• Sample preparation options:

  • Paraffin sections: Fix tissue in 4% paraformaldehyde, dehydrate, embed, and section (5-10μm)

  • Cryosections: Embed in OCT compound, freeze, and section (10-20μm)

  • Whole-mount preparations: For roots or other thin tissues, fix directly and process intact

• Optimized immunofluorescence protocol:

  • Fix samples in 4% paraformaldehyde in PBS (pH 7.4) for 30 minutes

  • Wash 3× with PBS (5 minutes each)

  • Permeabilize with 0.1% Triton X-100 in PBS (10 minutes)

  • Block with 3% BSA, 0.1% Triton X-100 in PBS (1 hour)

  • Incubate with At4g17200 antibody (1:200 dilution) in blocking buffer overnight at 4°C

  • Wash 5× with PBS containing 0.1% Triton X-100

  • Incubate with fluorescent secondary antibody (1:500) for 2 hours at room temperature

  • Counterstain with DAPI (1μg/ml, 10 minutes)

  • Mount in anti-fade medium

• Critical controls:

  • Secondary antibody-only control to assess non-specific binding

  • Pre-immune serum control at equivalent concentration

  • Autofluorescence control (untreated sample)

  • If available, At4g17200 knockout/knockdown tissue as negative control

• Autofluorescence management:

  • Treat sections with 0.1% sodium borohydride in PBS (10 minutes) before blocking

  • Consider CuSO₄ treatment (10mM in 50mM ammonium acetate, pH 5.0) for 30 minutes

  • Use far-red fluorophores for detection to minimize overlap with plant autofluorescence

  • Implement spectral unmixing during image acquisition if available

How can I integrate At4g17200 Antibody data with other -omics approaches?

Integrating antibody-based data with other -omics approaches provides a comprehensive understanding of At4g17200 function through complementary perspectives:

Table 2: Multi-omics Integration Strategy for At4g17200 Research

Implement this integration through:

• Experimental design consistency:

  • Use identical plant growth conditions across experiments

  • Sample at the same developmental stages

  • Apply consistent stress treatments when applicable

• Data analysis workflow:

  • Normalize datasets appropriately for cross-platform comparison

  • Apply multivariate statistical methods (PCA, cluster analysis)

  • Use network analysis tools like Cytoscape to visualize relationships

  • Consider machine learning approaches to identify non-obvious patterns

• Functional validation:

  • Test hypotheses generated from integrated analysis

  • Use genetic approaches (knockout/overexpression) to validate predictions

  • Employ pharmacological interventions when available

This systems biology approach can reveal functional insights about At4g17200 that might remain hidden when using any single technique.

How can I address common challenges with At4g17200 Antibody applications?

Researchers working with the At4g17200 antibody may encounter several technical challenges. The following systematic approaches can help resolve common issues:

Table 3: Troubleshooting Guide for At4g17200 Antibody Applications

For particularly challenging applications, consider:

• Using alternative detection methods (fluorescent vs. chemiluminescent)

• Comparing different tissue types or developmental stages for optimal protein expression

• Pre-adsorbing antibody with non-specific proteins to reduce background

• Employing protein concentration techniques for low-abundance targets

What epitope accessibility factors affect At4g17200 Antibody performance?

Epitope accessibility significantly impacts antibody performance across different experimental conditions. For the At4g17200 antibody, consider these influencing factors:

• Fixation effects:

  • Aldehyde fixatives can mask epitopes through protein cross-linking

  • Methanol fixation may better preserve certain epitopes by precipitating proteins without cross-linking

  • For critical applications, compare multiple fixation methods to determine optimal epitope preservation

• Denaturation considerations:

  • If the antibody was raised against a linear peptide, it may function optimally in Western blots with denatured proteins

  • For applications requiring native protein recognition (immunoprecipitation), verify antibody performance with native protein preparations

  • Consider the antibody's recognition of conformational versus linear epitopes

• Antigen retrieval methods for microscopy:

  • Heat-induced epitope retrieval (10mM sodium citrate buffer pH 6.0, 95°C for 15 minutes)

  • Enzymatic retrieval (proteinase K treatment, 20μg/ml for 10 minutes)

  • Test multiple methods in parallel to determine optimal epitope accessibility

• Sample processing effects:

  • Buffer composition can affect protein conformation and epitope accessibility

  • For membrane-associated proteins, detergent selection is critical (compare 0.1% Triton X-100, 0.5% NP-40)

  • For nuclear proteins, ensure adequate nuclear membrane permeabilization (0.5% Triton X-100 for 15 minutes)

• Post-translational modifications:

  • Phosphorylation, glycosylation, or other modifications may affect epitope recognition

  • When suspected, employ enzymatic treatments (phosphatases, glycosidases) before antibody application

  • Compare detection across different tissue types or conditions where modification states may vary

Systematic optimization of these parameters will maximize the utility of the At4g17200 antibody across diverse experimental applications.

How can the At4g17200 Antibody advance plant stress response studies?

The At4g17200 antibody offers valuable opportunities for investigating potential roles of the target protein in plant stress responses through multiple experimental approaches:

• Expression profiling under stress conditions:

  • Quantify At4g17200 protein levels during abiotic stress exposure (drought, salinity, temperature extremes, nutrient deficiency)

  • Monitor expression during pathogen infection or herbivore attack

  • Compare protein regulation across multiple stress types to identify specific or general stress responses

• Subcellular localization changes:

  • Track potential stress-induced relocalization using immunofluorescence microscopy

  • Compare control versus stressed samples to identify dynamic changes in protein localization

  • Correlate localization changes with onset of stress responses

• Protein interaction network modulation:

  • Use co-immunoprecipitation under control and stress conditions to identify stress-specific protein interactions

  • Verify interactions with reciprocal pulldowns and in vitro binding assays

  • Map interaction networks to understand functional relationships

• Post-translational modification analysis:

  • Immunoprecipitate At4g17200 protein from control and stressed tissues

  • Analyze via mass spectrometry for phosphorylation, ubiquitination, or other modifications

  • Correlate modifications with protein activity or localization changes

Since the function of At4g17200 remains uncharacterized, these approaches may provide the first insights into its biological role and significance in plant environmental adaptation.

What future research directions might expand At4g17200 Antibody applications?

Given the limited published research on the At4g17200 protein, several promising directions could expand antibody applications and contribute to understanding this uncharacterized protein:

• Comprehensive expression atlas development:

  • Map protein expression across tissues, developmental stages, and environmental conditions

  • Create standardized antibody-based detection protocols optimized for different plant tissues

  • Develop quantitative Western blot standards for comparing expression levels

• Functional genomics integration:

  • Generate tagged At4g17200 lines for dual antibody detection

  • Develop inducible knockdown/overexpression systems to manipulate protein levels

  • Correlate protein levels with phenotypic outcomes to infer function

• Evolutionary conservation studies:

  • Test antibody cross-reactivity across plant species

  • Compare recognition patterns with bioinformatic predictions of epitope conservation

  • Investigate functional conservation of homologous proteins across species

• Technical advancements:

  • Develop monoclonal antibodies against different epitopes of At4g17200

  • Create antibodies specific for potential post-translational modifications

  • Adapt super-resolution microscopy techniques for detailed localization studies

• Applied research applications:

  • Investigate At4g17200 regulation in crop species under agricultural conditions

  • Explore potential biotechnological applications if stress-responsive properties are confirmed

  • Develop high-throughput screening methods incorporating the antibody for mutant analysis

These directions would address the current knowledge gap regarding At4g17200 function while establishing new methodological approaches for plant protein research.