At1g60180 Antibody

What is At1g60180 and why are antibodies needed for its study?

At1g60180 is an Arabidopsis thaliana gene identifier representing a specific locus on chromosome 1. Antibodies against the protein encoded by this gene are essential tools for investigating its expression patterns, subcellular localization, and functional roles in plant development. Research demonstrates that antibodies enable better understanding of "protein localization at sub-cellular, cellular and tissue levels" which contributes significantly to elucidating "their function and role in cell and tissue dynamics, protein-protein interactions and protein regulatory networks" .

What approaches are most effective for generating antibodies against Arabidopsis proteins?

Two primary approaches exist for generating antibodies against Arabidopsis proteins, with significantly different success rates:

The recombinant protein approach has proven substantially more effective for Arabidopsis proteins. The poor performance of peptide antibodies may be attributed to epitope prediction challenges, as "prediction methods identify individual stretches of amino acids (continuous epitopes), whereas epitopes are very often discontinuous, involving distant subsequences brought together by the protein's tertiary structures" .

How should researchers select between polyclonal and monoclonal antibodies for plant protein studies?

Both antibody types have distinct advantages for plant research:

The majority of Arabidopsis antibodies in repositories are polyclonal due to their higher sensitivity and easier production, but researchers should consider experimental requirements and specificity needs when selecting antibody type.

What experimental techniques can utilize At1g60180 antibody effectively?

Antibodies against plant proteins can be employed in diverse experimental techniques, each requiring specific optimization:

When establishing protocols for At1g60180 antibody, researchers should empirically determine optimal conditions for each application through titration experiments.

How can researchers validate antibody specificity for At1g60180?

Multiple validation approaches should be combined to ensure antibody specificity:

• Genetic validation using mutants: "The specificity of the ADL1Ap-specific antibodies and the identity of the 68-kD polypeptide as ADL1Ap were confirmed by demonstrating that homozygous adl1A mutant seedlings lack the 68-kD protein" . This represents the gold standard for antibody validation.

• Bioinformatic analysis: "Bioinformatic analysis was used to identify potential antigenic regions and then the largest antigenic subsequence was checked for potential cross-reactivity by database searches using blastX" . A similarity score cutoff of 40% at amino acid level is recommended as a threshold for antigenic region selection.

• Multiple antibody comparison: Using "a combination of antibodies specific for the GTPase domain or a ADL1Ap-specific peptide" can strengthen confidence in localization results.

• Affinity purification: "Affinity purification of antibodies massively improved the detection rate" , reducing background and enhancing specific signal.

What controls are essential for At1g60180 antibody experiments?

Implementing these controls is critical for establishing confidence in At1g60180 antibody results and avoiding misinterpretation of experimental data.

How can researchers overcome common pitfalls with plant protein antibodies?

Several technical challenges frequently arise when working with plant protein antibodies:

How does affinity purification improve antibody performance?

Affinity purification dramatically enhances antibody quality through several mechanisms:

The multi-step purification process typically involves:

• Coupling the antigen to an affinity matrix

• Applying crude serum to the column

• Washing away non-specific antibodies

• Eluting and concentrating specific antibodies

• Buffer exchange and quality control testing

What buffer conditions are optimal for At1g60180 antibody applications?

While specific conditions for At1g60180 antibody are not directly provided in the search results, optimal buffer conditions for Arabidopsis protein antibodies generally include:

For Western blotting:

• Extraction buffer: "20 mM Tris pH 7.5, 5 mM MgCl₂, 2.5 mM DTT, 300 mM NaCl, 0.1% NP-40, 1% proteasome inhibitor MG132"

• Blocking solution: "5% low-fat milk powder in TBS-TT (0.25% TWEEN20; 0.1% Triton-X)"

• Antibody dilution buffer: "TBS-TT containing 5% low fat milk powder"

For immunocytochemistry:

• Fixation conditions: Typically 4% paraformaldehyde in PBS

• Permeabilization: 0.1-0.5% Triton X-100

• Blocking: 2-5% BSA or normal serum

Buffer optimization should be performed empirically for each new antibody, as small adjustments can significantly improve signal quality.

How can At1g60180 antibody be integrated with genetic studies?

Combining antibody-based techniques with genetic approaches provides powerful insights:

• Protein localization in mutant backgrounds: Antibodies allow visualization of how protein distribution changes in various genetic backgrounds. This approach was demonstrated with LAX2 antibody, which "detected a strong signal in the root apex in wild type Columbia roots but not in null lax2 mutants" .

• Functional redundancy assessment: By comparing protein levels of related family members in single mutants, researchers can identify compensatory mechanisms. For example, "Phenotypic analysis of these loss-of-function mutant plants suggests that other members of the ADL1 gene family have functions that partially compensate for the loss of ADL1Ap function" .

• Protein complex analysis: Antibodies can help determine if At1g60180 protein exists in complexes with other proteins and how these interactions change in different genetic backgrounds.

• Developmental expression studies: Combining antibody detection with developmental stage-specific or tissue-specific mutants can reveal complex regulation patterns.

What approaches exist for studying protein-protein interactions involving At1g60180?

Several antibody-dependent techniques can reveal protein-protein interactions:

When designing such experiments, researchers should consider that "better understanding of protein localization at sub-cellular, cellular and tissue levels is likely to result in better understanding of their function and role in cell and tissue dynamics, protein–protein interactions and protein regulatory networks" .

How can quantitative proteomics be combined with At1g60180 antibody studies?

Integrating antibody techniques with quantitative proteomics offers several advantages:

• Immunoprecipitation followed by mass spectrometry (IP-MS): Using At1g60180 antibodies to enrich for the target protein and its interacting partners before mass spectrometric analysis.

• Selected Reaction Monitoring (SRM) validation: Antibody-based detection can validate findings from SRM-based quantitative proteomics.

• Parallel Reaction Monitoring (PRM): Combining antibody-based fractionation with targeted mass spectrometry for enhanced sensitivity.

• Absolute quantification: Using purified standards and antibody-based detection for absolute protein quantification.

How should researchers interpret conflicting antibody results from different studies?

When faced with conflicting results:

• Evaluate antibody specificity: "This GTPase domain has been identified in several other genes in the Arabidopsis genome that are predicted to encode 68-kD dynamin-like proteins. This calls into question the specificity of the antibodies used in these studies" .

• Consider technical differences: Examine fixation methods, extraction buffers, antibody dilutions, and detection systems.

• Validate with multiple approaches: Combine different techniques (WB, IF, IP) and use multiple antibodies against different epitopes.

• Incorporate genetic validation: "The specificity of the ADL1Ap-specific antibodies and the identity of the 68-kD polypeptide as ADL1Ap were confirmed by demonstrating that homozygous adl1A mutant seedlings lack the 68-kD protein" .

What impact do epitope accessibility issues have on At1g60180 antibody effectiveness?

Epitope accessibility significantly affects antibody performance across different applications:

• Challenges with peptide antibodies: "The prediction methods identify individual stretches of amino acids (continuous epitopes), whereas epitopes are very often discontinuous, involving distant subsequences brought together by the protein's tertiary structures" .

• Native vs. denatured protein recognition: "A synthetic continuous (or even discontinuous epitope) peptide may still not fold correctly and hence not generate antibodies that recognize the native protein structure" .

• Technique-specific considerations:

  • Western blotting: Denatured proteins expose linear epitopes

  • Immunofluorescence: Fixed proteins maintain some tertiary structure

  • Immunoprecipitation: Requires antibodies recognizing native conformations

Researchers should evaluate At1g60180 antibody performance in each specific application rather than assuming uniform effectiveness across techniques.

How can researchers assess antibody batch-to-batch variation and its impact on experimental reproducibility?

Antibody variation between batches is a significant concern for experimental reproducibility:

When working with At1g60180 antibody across multiple studies or time periods, researchers should maintain detailed records of antibody batch information and include standardized positive controls in each experiment to enable accurate cross-study comparisons.