At2g07140 Antibody

What is the At2g07140 protein and why is it studied?

The At2g07140 protein (UniProt accession: Q9ZV78) is a protein expressed in Arabidopsis thaliana, commonly known as Mouse-ear cress. This protein is the target of the At2g07140 antibody (catalog number CSB-PA154198XA01DOA) . Arabidopsis thaliana serves as an important model organism in plant biology research due to its relatively small genome, short generation time, and well-characterized genetics. The At2g07140 protein is studied to understand its specific cellular functions, expression patterns across different tissues, and its potential role in plant development or stress responses.

When designing experiments to study this protein, researchers should consider:

• The protein's predicted molecular weight and expected cellular localization

• Known expression patterns across different plant tissues and developmental stages

• Potential post-translational modifications that might affect antibody binding

• Available genetic resources such as knockout or overexpression lines in Arabidopsis

How should I validate the specificity of the At2g07140 antibody?

Antibody validation is critical for ensuring experimental reproducibility and reliability. Following the enhanced validation approach described in current literature, you should employ multiple orthogonal methods to validate the At2g07140 antibody . A comprehensive validation strategy includes:

• Western blot analysis to confirm the antibody detects a protein of the expected molecular weight

• Comparison with RNA expression data to verify consistency between protein and transcript levels

• Use of genetic knockouts or knockdowns as negative controls

• Testing in multiple relevant tissues where the protein is expected to be expressed

• When possible, validation with independent antibodies targeting different epitopes of the same protein

According to established validation criteria, an antibody can be considered "approved" when it shows RNA expression pattern consistency and when the staining pattern is consistent with available literature, or when paired antibodies show similar expression patterns .

What controls should I include in experiments using the At2g07140 antibody?

Proper experimental controls are essential for interpreting results obtained with the At2g07140 antibody. Include the following controls:

• Positive control: A tissue or sample known to express the target protein

• Negative control:

  • Primary antibody omission

  • Tissue from knockout/knockdown plants (if available)

  • Pre-absorption with the immunizing peptide/antigen

• Loading control: Detection of a housekeeping protein to normalize expression levels

• Isotype control: Use of an irrelevant antibody of the same isotype

Including these controls helps distinguish specific from non-specific binding and provides benchmark comparisons to validate experimental findings.

How do I troubleshoot weak or absent signal when using the At2g07140 antibody?

When facing weak or absent signals with the At2g07140 antibody, systematically investigate the following factors:

• Antibody concentration: Titrate the antibody to determine optimal concentration

• Antigen retrieval (for immunohistochemistry): Test multiple retrieval methods

• Incubation conditions: Adjust time, temperature, and buffer composition

• Blocking reagents: Test different blocking solutions to reduce background while maintaining specific signal

• Detection system sensitivity: Consider enzyme-based vs. fluorescence-based detection

• Sample preparation: Ensure protein extraction methods preserve the epitope

• Target protein expression level: Verify if the target protein is expressed in your samples

Remember that antibody responses can be transient or variable in certain conditions, as observed in other biological systems . If your target protein is expressed at low levels, consider using amplification methods or more sensitive detection reagents.

How can I determine if cross-reactivity is affecting my experimental results?

Cross-reactivity occurs when an antibody binds to proteins other than its intended target. To assess and address cross-reactivity with the At2g07140 antibody:

• Perform western blot analysis and look for unexpected bands

• Compare immunostaining patterns with known expression patterns from RNA data

• Test the antibody in tissues known not to express the target

• Use mass spectrometry for immunoprecipitated samples to identify all bound proteins

• Test the antibody in different species to evaluate conservation-based cross-reactivity

The enhanced validation criteria emphasize the importance of RNA similarity scores in determining antibody specificity . An antibody with high or medium RNA consistency scores is more likely to be specific to its target.

What advanced approaches can I use to validate At2g07140 antibody for spatial protein analysis?

For rigorous spatial protein analysis using the At2g07140 antibody, consider implementing these advanced validation approaches:

• Orthogonal validation: Compare results from antibody-based detection with alternative methods such as mRNA in situ hybridization or reporter gene fusions

• Independent antibodies: Use multiple antibodies targeting different epitopes of the At2g07140 protein

• Genetic approaches: Compare wildtype with knockout/knockdown plants

• Super-resolution microscopy: For precise subcellular localization, combined with co-localization studies with known organelle markers

• Proximity ligation assays: To validate protein-protein interactions

These approaches align with the enhanced validation strategy that has successfully uncovered missing proteins and proteins of unknown function in human tissues .

What is the optimal protocol for immunohistochemistry using the At2g07140 antibody?

For optimal immunohistochemistry results with the At2g07140 antibody in plant tissues:

• Tissue fixation:

  • Use 4% paraformaldehyde for 24 hours

  • Alternatively, test zinc-based fixatives which may better preserve some epitopes

• Sectioning:

  • For paraffin sections: 5-8 μm thickness

  • For frozen sections: 10-15 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval: Citrate buffer (pH 6.0), 95°C for 20 minutes

  • Enzymatic retrieval: Proteinase K (20 μg/ml) for 10-15 minutes

• Blocking:

  • 5% normal serum (from the species in which the secondary antibody was raised)

  • 1% BSA in PBS with 0.1% Triton X-100

  • Block for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: Start with 1:100-1:500 (optimize as needed)

  • Incubate overnight at 4°C

• Detection system:

  • For brightfield: HRP/AP-based detection systems

  • For fluorescence: Use fluorophore-conjugated secondary antibodies

• Counterstaining:

  • DAPI for nuclei

  • Calcofluor white for cell walls

This protocol should be optimized based on specific tissue types and experimental requirements.

How do I select between different antibody-based techniques for studying At2g07140?

The choice of technique depends on your specific research question:

When selecting a technique, consider:

• The level of sensitivity required

• Whether spatial information is important

• If you need quantitative or qualitative data

• Available equipment and expertise

How can I adapt antibody-based techniques for different Arabidopsis tissue types?

Different Arabidopsis tissues require specific adaptations for optimal antibody performance:

• Leaf tissue:

  • Challenge: High chlorophyll autofluorescence

  • Adaptation: Use brightfield IHC or red-shifted fluorophores; pre-treat samples with sodium borohydride to reduce autofluorescence

• Root tissue:

  • Challenge: Dense cell walls

  • Adaptation: Extended proteinase K digestion; consider vibratome sectioning for thicker samples

• Seed/silique:

  • Challenge: Hard tissues, difficult penetration

  • Adaptation: Extended fixation; additional permeabilization steps; vacuum infiltration

• Floral tissue:

  • Challenge: Complex morphology, variable protein expression

  • Adaptation: Careful orientation during embedding; modified clearing protocols

• Embryos:

  • Challenge: Small size, difficult to manipulate

  • Adaptation: Whole-mount procedures; extended antibody incubation times

For all tissue types, consider using the bispecific antibody design principles discussed in current literature to enhance specificity and reduce background .

How do I quantify At2g07140 protein expression levels from immunohistochemistry data?

For quantitative analysis of immunohistochemistry data:

• Image acquisition:

  • Use consistent exposure settings across all samples

  • Capture multiple fields per sample (minimum 5-10)

  • Include all relevant controls in the same imaging session

• Software-based quantification:

  • Use ImageJ/Fiji with appropriate plugins

  • Define regions of interest (ROIs) consistently across samples

  • Measure parameters such as:

    • Mean signal intensity

    • Integrated density

    • Area percentage

    • Colocalization coefficients (if applicable)

• Normalization:

  • Subtract background signal

  • Normalize to internal controls

  • Consider cell/tissue density variations

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution

  • Compare between experimental groups

  • Report both statistical significance and effect size

Remember that antibody-based quantification provides relative rather than absolute values and should be interpreted accordingly.

How should I interpret discrepancies between antibody results and transcript data for At2g07140?

When facing discrepancies between antibody-detected protein levels and transcript data:

• Consider post-transcriptional regulation:

  • microRNA-mediated suppression

  • Translation efficiency differences

  • Protein stability and turnover rates

• Evaluate technical factors:

  • Antibody specificity issues

  • Detection method sensitivity thresholds

  • RNA quantification accuracy

• Biological explanations:

  • Temporal delays between transcription and translation

  • Tissue-specific post-transcriptional regulation

  • Protein transport between tissues

• Validation approaches:

  • Use orthogonal protein detection methods

  • Apply enhanced validation criteria as described in current literature

  • Test in multiple experimental systems

The RNA similarity score method described in enhanced validation approaches can help determine if such discrepancies represent a true biological phenomenon or a technical artifact .

How can the At2g07140 antibody be used in multi-omics research approaches?

Integrating the At2g07140 antibody into multi-omics research:

• Proteomics integration:

  • Use the antibody for immunoprecipitation followed by mass spectrometry

  • Identify interaction partners and post-translational modifications

  • Compare with predicted protein-protein interaction networks

• Transcriptomics correlation:

  • Map protein expression against transcriptome data

  • Identify discordant regions suggesting post-transcriptional regulation

  • Use single-cell approaches to resolve cell-type specific variations

• Metabolomics connections:

  • Connect protein localization with metabolite distributions

  • Identify potential metabolic roles of At2g07140

  • Correlate protein levels with metabolic pathway activities

• Phenomics relationships:

  • Link protein expression patterns with phenotypic variations

  • Use in genetic screens to identify functional relationships

  • Apply in natural variation studies

This multi-omics approach provides a comprehensive understanding of At2g07140 function within the broader biological context of Arabidopsis thaliana.

What considerations are important when developing new antibodies against At2g07140 or related proteins?

When developing new antibodies against At2g07140 or related proteins:

• Epitope selection:

  • Choose regions with low sequence similarity to other proteins

  • Avoid transmembrane domains and highly conserved functional domains

  • Consider multiple epitopes for comprehensive protein detection

• Antibody format considerations:

  • Evaluate the advantages of different formats (polyclonal, monoclonal, recombinant)

  • Consider bispecific antibody approaches for enhanced specificity

  • Assess the need for symmetric versus asymmetric binding

• Validation strategy planning:

  • Design validation experiments before antibody production

  • Include orthogonal methods for validation

  • Prepare appropriate positive and negative controls

• Production optimization:

  • Consider the expression system impact on post-translational modifications

  • Evaluate purification strategies to maintain epitope integrity

  • Test different conjugation approaches for specialized applications

• Documentation:

  • Record detailed validation data for reproducibility

  • Document experimental conditions affecting performance

  • Share validation data with the research community

These considerations align with modern antibody engineering principles that emphasize both functionality and developability .