At2g29600 Antibody

What is At2g29600 and why is it studied in plant biology?

At2g29600 is a gene locus in Arabidopsis thaliana that encodes a specific protein with Uniprot accession number Q9ZW38. This protein is studied primarily in plant molecular biology research to understand its function in plant development and stress responses. The antibody against this protein enables researchers to detect, localize, and quantify the At2g29600 protein expression in various experimental conditions . The importance of this protein stems from its potential role in signaling pathways, making it valuable for understanding fundamental plant biological processes.

What are the validated experimental applications for At2g29600 antibody?

The At2g29600 antibody has been validated for enzyme-linked immunosorbent assay (ELISA) and Western blotting (WB) applications . These techniques enable researchers to detect and quantify the At2g29600 protein in plant tissue samples. The antibody's specific binding to the target protein makes it suitable for analyzing protein expression levels across different experimental conditions, developmental stages, or in response to various stimuli. When designing experiments, researchers should optimize protocols specifically for plant tissue samples, as extraction conditions may differ from those used with animal tissues.

What storage conditions maximize At2g29600 antibody stability and shelf life?

For optimal stability and performance, the At2g29600 antibody should be stored at -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and reduce binding efficiency. The antibody is supplied in liquid form in a preservative buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4 . This formulation is designed to maintain antibody integrity during proper storage. For working solutions, researchers should prepare small aliquots sufficient for single experiments to minimize freeze-thaw cycles of the stock solution.

How should negative and positive controls be designed for At2g29600 antibody experiments?

For robust experimental design with At2g29600 antibody, proper controls are essential. For positive controls, researchers should use wild-type Arabidopsis thaliana samples where At2g29600 protein is known to be expressed. For negative controls, consider using:

• Samples from At2g29600 knockout mutants (if available)

• Pre-immune serum at the same concentration as the primary antibody

• Secondary antibody-only controls (omitting the primary antibody)

• Non-plant samples or plant species with significant divergence from Arabidopsis thaliana

Each control should be processed identically to experimental samples. In Western blot applications, successful positive controls should show a clear band at the expected molecular weight of the At2g29600 protein, while negative controls should show no specific binding .

What optimization strategies improve At2g29600 antibody performance in Western blotting?

For optimal Western blot results with At2g29600 antibody, consider these technical optimization strategies:

Additionally, consider using specialized plant protein extraction buffers containing protease inhibitors to prevent degradation of the target protein. The antigen affinity purification method used for this antibody suggests high specificity, but optimization remains important for each experimental system.

What are the key considerations for cross-reactivity testing with At2g29600 antibody?

Cross-reactivity assessment is crucial when working with antibodies in plant systems. For At2g29600 antibody, consider these methodological approaches:

• Perform sequence alignment of At2g29600 with homologous proteins from other species

• Test the antibody on protein extracts from multiple plant species with varying evolutionary distances from Arabidopsis

• Include knockout or knockdown controls where the target protein is absent or reduced

• Conduct peptide competition assays by pre-incubating the antibody with excess immunizing peptide

How can At2g29600 antibody be employed in protein-protein interaction studies?

At2g29600 antibody can be effectively utilized in protein-protein interaction studies through these methodological approaches:

• Co-immunoprecipitation (Co-IP): Use the antibody to pull down At2g29600 protein complexes from plant lysates, followed by mass spectrometry to identify interacting partners

• Proximity ligation assay (PLA): Combine At2g29600 antibody with antibodies against suspected interacting proteins to visualize protein interactions in situ

• Chromatin immunoprecipitation (ChIP): If At2g29600 functions in transcriptional regulation, ChIP can identify DNA binding sites

• Yeast two-hybrid screening validation: Confirm interactions identified in screening by Co-IP with the antibody

For Co-IP applications, researchers should optimize lysis conditions to preserve native protein complexes while ensuring efficient extraction from plant tissues. Cross-linking approaches may be necessary to capture transient or weak interactions. The polyclonal nature of this antibody provides an advantage in recognizing multiple epitopes, potentially preserving antibody recognition even when some binding sites are obscured by protein-protein interactions.

What approaches enable quantitative analysis of At2g29600 protein levels across experimental conditions?

For precise quantification of At2g29600 protein levels, consider these methodological approaches:

• Quantitative Western blotting:

  • Use internal loading controls (housekeeping proteins)

  • Employ fluorescent secondary antibodies for wider linear dynamic range

  • Analyze with densitometry software using standard curves

• ELISA-based quantification:

  • Develop a sandwich ELISA using the At2g29600 antibody

  • Create standard curves with recombinant At2g29600 protein

  • Optimize sample preparation to minimize matrix effects

• Quantitative immunohistochemistry:

  • Use consistent staining protocols and imaging parameters

  • Include calibration standards in each experiment

  • Apply digital image analysis for fluorescence intensity quantification

The antibody has been validated for both Western blotting and ELISA applications , making these approaches particularly suitable. When designing quantitative experiments, consider the antibody's affinity, the abundance of the target protein, and the sensitivity of detection methods.

How can modern structural biology approaches be integrated with At2g29600 antibody research?

Integration of structural biology approaches with At2g29600 antibody research can provide deeper insights into protein function:

• Epitope mapping: Identify the specific binding regions of the antibody using:

  • Peptide arrays spanning the At2g29600 sequence

  • Hydrogen-deuterium exchange mass spectrometry

  • X-ray crystallography of antibody-antigen complexes

• Conformational studies:

  • Use the antibody to detect structural changes in At2g29600 under different conditions

  • Combine with circular dichroism or fluorescence spectroscopy

• Cryo-EM applications:

  • Utilize the antibody to stabilize protein complexes for structural determination

  • Image specific subcellular localizations with immunogold labeling

Recent advances in computational antibody design, as demonstrated in contemporary research, indicate that structural information can significantly enhance antibody specificity and affinity . The recombinant Arabidopsis thaliana At2g29600 protein used as the immunogen suggests potential for structural studies, as recombinant proteins can be engineered for structural analysis.

What are common sources of false positives/negatives when using At2g29600 antibody, and how can they be addressed?

When working with At2g29600 antibody, several technical issues can lead to misleading results:

Sources of false positives:

• Cross-reactivity with homologous proteins

• Non-specific binding to denatured proteins or hydrophobic regions

• Overly sensitive detection systems (excessive exposure)

• Contamination of secondary antibodies

Sources of false negatives:

• Insufficient protein extraction from plant tissues

• Epitope masking due to protein modifications or interactions

• Protein degradation during sample preparation

• Antibody deterioration from improper storage

Methodological solutions:

• Validate antibody specificity using knockout controls or competing peptides

• Optimize blocking conditions (5% BSA or milk can reduce non-specific binding)

• Include appropriate positive controls in every experiment

• Store antibody according to manufacturer recommendations (-20°C or -80°C, avoiding freeze-thaw cycles)

• Use fresh protease inhibitors in extraction buffers

• Consider native vs. denaturing conditions based on epitope accessibility

The polyclonal nature of this antibody may provide advantages in detecting the target under various conditions, as multiple epitopes are recognized, reducing the risk of complete signal loss due to a single epitope modification.

How should researchers interpret unexpected molecular weight variations of At2g29600 in Western blots?

When At2g29600 appears at unexpected molecular weights in Western blots, consider these interpretations and verification approaches:

Potential causes of higher molecular weight:

• Post-translational modifications (phosphorylation, glycosylation, ubiquitination)

• Incomplete denaturation leading to oligomerization

• Protein complexes resistant to SDS treatment

Potential causes of lower molecular weight:

• Proteolytic degradation during sample preparation

• Alternative splicing variants

• Protein processing/maturation in vivo

Verification methodologies:

• Treatment with specific enzymes:

  • Phosphatases for phosphorylated proteins

  • Glycosidases for glycosylated proteins

  • Deubiquitinating enzymes for ubiquitinated proteins

• Vary sample preparation conditions:

  • Use different detergents or chaotropic agents

  • Test multiple reducing agent concentrations

  • Vary heating conditions (time and temperature)

• Compare results across different tissues or developmental stages

The specific molecular weight of the At2g29600 protein should be verified experimentally and compared with theoretical predictions based on its amino acid sequence. The antibody is specifically generated against recombinant Arabidopsis thaliana At2g29600 protein , providing a reference point for expected molecular weight.

What statistical approaches are recommended for analyzing quantitative data from At2g29600 antibody experiments?

For robust statistical analysis of quantitative data from At2g29600 antibody experiments:

Experimental design considerations:

• Include at least 3-5 biological replicates

• Perform technical replicates for each biological sample

• Randomize sample processing order to minimize batch effects

• Include appropriate controls in each experimental batch

Statistical analysis approaches:

• For comparing two conditions:

  • Student's t-test (parametric data)

  • Mann-Whitney U test (non-parametric data)

• For multiple conditions:

  • One-way ANOVA with post-hoc tests (Tukey's or Bonferroni)

  • Kruskal-Wallis test for non-parametric data

• For complex experimental designs:

  • Two-way ANOVA for testing multiple factors

  • Mixed-effects models for repeated measures designs

• Data visualization:

  • Box plots showing distribution of values

  • Individual data points alongside means/medians

  • Error bars representing standard deviation or standard error

For Western blot densitometry, always normalize to loading controls and consider the non-linear nature of film-based detection methods. For ELISA data, use appropriate standard curves and consider testing for parallelism when analyzing complex samples.

How does At2g29600 antibody performance compare in diverse plant tissue types and preparation methods?

The performance of At2g29600 antibody can vary significantly across different plant tissues and preparation methods:

Tissue-specific considerations:

• Leaf tissue: Generally straightforward extraction, but secondary metabolites may interfere

• Root tissue: May require more stringent extraction conditions

• Reproductive tissues: Often contain unique proteins that may cross-react

• Seed tissue: High protein and lipid content can interfere with extraction and detection

Optimized preparation methods by tissue type:

For all tissue types, adding DTT (1-5mM) and a complete protease inhibitor cocktail is recommended. The antibody's affinity purification method suggests it should perform well across various sample types, but empirical optimization remains essential for each tissue type.

How can At2g29600 antibody be incorporated into high-throughput screening applications?

To incorporate At2g29600 antibody into high-throughput screening applications, consider these methodological approaches:

• Automated ELISA platforms:

  • Develop a sandwich ELISA format using At2g29600 antibody

  • Optimize for 96- or 384-well plate formats

  • Establish robust positive and negative controls

  • Implement liquid handling robots for consistent pipetting

• Protein array applications:

  • Spot candidate interacting proteins on arrays

  • Probe with At2g29600 protein followed by the antibody detection

  • Alternatively, use the antibody to detect At2g29600 binding to arrayed proteins

• Automated Western blotting systems:

  • Implement capillary-based Western platforms

  • Optimize antibody dilution for reduced consumption

  • Develop quantitative analysis workflows

• High-content imaging:

  • Use fluorescently-tagged secondary antibodies for microscopy

  • Employ automated image analysis for quantification

  • Incorporate machine learning for complex phenotype identification

For high-throughput applications, larger quantities of antibody may be required, so scalability of production is an important consideration. The polyclonal nature of this antibody may provide robust detection across various platform conditions, though batch-to-batch variability should be monitored.

What novel computational approaches can enhance data interpretation from At2g29600 antibody experiments?

Advanced computational methods can significantly enhance the value of data generated from At2g29600 antibody experiments:

• Network analysis approaches:

  • Integrate At2g29600 interaction data with publicly available protein interaction databases

  • Apply graph theory algorithms to identify functional modules

  • Use centrality measures to assess the importance of At2g29600 in protein networks

• Machine learning for Western blot analysis:

  • Train convolutional neural networks to detect and quantify bands

  • Develop algorithms to normalize across multiple blots

  • Implement automated quality control for image analysis

• Multiomics data integration:

  • Correlate protein levels (detected by antibody) with transcriptomics data

  • Integrate with metabolomics data to identify functional relationships

  • Apply dimensionality reduction techniques (PCA, t-SNE) to visualize complex relationships

• Structure-based computational approaches:

  • Model the interaction between the antibody and At2g29600 protein

  • Predict effects of amino acid variations on antibody binding

  • Apply molecular dynamics simulations to understand binding kinetics

Recent advances in de novo antibody design using computational approaches demonstrate the power of structure-based methods in antibody research. These computational tools can help researchers maximize the information obtained from At2g29600 antibody experiments and place findings in broader biological contexts.