ATL61 Antibody

What is ATL61 and what is its function in plant biology?

ATL61 (AT3G14320) is a putative RING-H2 finger protein found in Arabidopsis species, including Arabidopsis thaliana and Arabidopsis lyrata. This protein belongs to the RING-finger family, which typically functions in ubiquitin-mediated protein degradation pathways .

RING-H2 finger proteins like ATL61 contain zinc-binding domains and often serve as E3 ubiquitin ligases in plants, playing crucial roles in various cellular processes including stress responses, hormone signaling, and developmental regulation. The gene is classified as protein-coding and has been mapped with the Entrez Gene ID 820652 in Arabidopsis thaliana and 9321100 in Arabidopsis lyrata subspecies lyrata .

The protein's full sequence information is available, with the coding sequence spanning 579 base pairs as documented in reference sequences such as XM_002884988.1 . Understanding this basic molecular information is essential for researchers designing experiments to study ATL61's function or developing appropriate controls for antibody-based detection systems.

What experimental applications are supported by ATL61 antibody?

ATL61 antibody has been validated for several key experimental applications in plant biology research:

• Western Blotting (WB): The antibody can be used for detecting ATL61 protein expression in plant tissue lysates, allowing researchers to quantify relative protein levels under different experimental conditions .

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of ATL61 protein in solution, the antibody has been validated for ELISA applications .

The antibody specifications include:

The polyclonal nature of commercially available ATL61 antibodies means they recognize multiple epitopes on the target protein, potentially increasing detection sensitivity but requiring thorough validation to ensure specificity . When designing experiments, researchers should account for the antibody's specific characteristics to optimize detection conditions and appropriate controls.

How should ATL61 antibody validation be approached following current standards?

Antibody validation is essential for ensuring experimental reproducibility. For ATL61 antibody, researchers should follow the "five pillars" approach to validation recommended by the International Working Group for Antibody Validation :

• Genetic strategy: This is particularly important for ATL61 research. Using knockout or knockdown Arabidopsis lines lacking the ATL61 gene provides the strongest control for antibody specificity. The absence of signal in these genetic models compared to wild-type provides compelling evidence of specificity .

• Orthogonal strategy: Correlate protein detection using the ATL61 antibody with mRNA expression levels determined by RT-qPCR or RNA-seq. This validates that the antibody signal corresponds with gene expression patterns .

• Independent antibody strategy: Compare results using different antibodies targeting distinct epitopes of ATL61, if available. Concordant results strongly support antibody specificity .

• Recombinant expression strategy: Overexpress tagged versions of ATL61 in plant systems to confirm antibody detection of increased protein levels .

• Immunocapture MS strategy: Perform immunoprecipitation with ATL61 antibody followed by mass spectrometry to confirm the identity of captured proteins .

It's essential to document that: "the antibody is binding to the target protein; that the antibody binds to the target protein when in a complex mixture of proteins; that the antibody does not bind to proteins other than the target protein; that the antibody performs as expected in the experimental conditions used in the specific assay employed" . These validation steps should be performed in the specific experimental context where the antibody will be used.

What storage and handling conditions maintain ATL61 antibody performance?

Proper storage and handling of ATL61 antibody is critical for maintaining its functionality and ensuring experimental reproducibility:

• Temperature: Store ATL61 antibody at -20°C or -80°C for long-term stability . Multiple freeze-thaw cycles should be avoided as they can degrade antibody quality and affect binding efficiency.

• Shipping conditions: When ordering or transporting, the antibody should be maintained on blue ice to preserve activity .

• Working aliquots: Prepare small working aliquots of the antibody to avoid repeated freeze-thaw cycles of the entire stock.

• Positive control handling: The antibody may come with recombinant immunogen protein (200μg) that serves as a positive control; this should be stored according to the same temperature guidelines .

• Pre-immune serum: Properly store the pre-immune serum (if provided) as it serves as an important negative control for validating specificity .

Researchers should document any deviations from recommended storage conditions, as these can affect experimental outcomes and reproducibility. Antibody functionality should be periodically tested using positive controls, especially after extended storage periods.

How can ATL61 antibody be used to investigate plant stress response pathways?

ATL61 antibody can be a powerful tool for investigating the role of RING-H2 finger proteins in plant stress responses, which often involve ubiquitin-mediated protein degradation pathways. To effectively use ATL61 antibody in stress response studies:

• Time-course experiments: Monitor ATL61 protein levels using Western blotting at different timepoints following exposure to stressors (e.g., drought, salt, pathogens). This approach can reveal how quickly ATL61 responds to specific stresses and how long the response persists.

• Subcellular localization changes: Use immunofluorescence with ATL61 antibody to track potential changes in protein localization under stress conditions, which may indicate functional shifts.

• Protein-protein interaction studies: Employ co-immunoprecipitation with ATL61 antibody to identify stress-specific interaction partners, potentially revealing the substrates targeted by this putative E3 ligase under different conditions.

• Post-translational modification analysis: Combine ATL61 immunoprecipitation with mass spectrometry to detect stress-induced modifications of the protein itself, which might regulate its activity.

When designing these experiments, it's critical to remember that antibody performance can be context-dependent, and characterization needs to be performed by end users for each specific experimental application . Additionally, results may be cell or tissue type specific, necessitating validation across different plant tissues or developmental stages.

What are the challenges and solutions in optimizing Western blotting with ATL61 antibody?

Western blotting with ATL61 antibody presents several technical challenges that researchers should address:

• Cross-reactivity concerns: As a polyclonal antibody, ATL61 antibody may potentially recognize related RING-H2 finger proteins. To address this:

  • Include knockout/knockdown controls when possible

  • Use recombinant ATL61 as a positive control to identify the correct band size

  • Compare band patterns with RNA expression data using orthogonal methods

• Signal optimization: RING-H2 finger proteins may be expressed at low levels under basal conditions:

  • Optimize protein extraction using buffers containing appropriate protease inhibitors

  • Consider enrichment steps prior to loading samples

  • Test different blocking agents to reduce background while preserving specific signal

• Band verification protocol:

• Quantification considerations: When quantifying ATL61 expression changes:

  • Select appropriate housekeeping proteins for normalization that are stable under your experimental conditions

  • Use technical replicates to account for blotting variability

  • Employ statistical analysis appropriate for semi-quantitative data

Remember that "antibody specificity [is] 'context-dependent'" and characterization should be performed for each specific experimental condition .

How can computational tools enhance ATL61 antibody-based research?

Advanced computational tools are transforming antibody-based research and can specifically enhance studies utilizing ATL61 antibody:

• Epitope prediction: Computational algorithms can predict which regions of ATL61 are likely recognized by the polyclonal antibody, helping researchers understand potential cross-reactivity with related proteins. These predictions can guide experimental design, particularly when working with RING-H2 finger protein family members with structural similarity.

• AI-assisted antibody analysis: New deep learning approaches similar to those described for COVID-19 antibodies can be adapted for plant research. For example, the AF2Complex tool uses "deep learning to predict which antibodies could bind to [specific] protein[s]" . This approach could potentially:

  • Predict optimal antibody-antigen interactions for ATL61

  • Identify potential cross-reactivity with other plant proteins

  • Help design better experiments using existing antibodies

• Automated image analysis: For immunohistochemistry or immunofluorescence applications with ATL61 antibody, machine learning algorithms can:

  • Quantify staining patterns objectively

  • Identify subtle subcellular localization changes

  • Process large datasets consistently

• Integrative data analysis: Computational approaches can integrate antibody-derived data with:

  • Transcriptomic data to correlate protein and mRNA levels

  • Proteomic datasets to place ATL61 in functional networks

  • Phylogenetic information to understand conservation across species

Georgia Tech researchers demonstrated that by "combin[ing] deep learning with advanced sequencing techniques to predict how antibodies interact with antigens," they achieved "90% accuracy in predicting the best antibodies" . Similar approaches could be adapted for plant antibodies like ATL61 to enhance experimental design and interpretation.

What strategies address antibody batch variability issues in long-term ATL61 research?

Antibody batch variability is a significant challenge for longitudinal studies. For ATL61 research spanning extended periods, consider implementing these strategies:

• Standardization with recombinant controls: Use recombinant ATL61 protein as a standard reference across different antibody lots. By normalizing signals to this control, researchers can account for sensitivity differences between batches.

• Batch validation protocol:

• Transition to recombinant antibodies: Consider developing or sourcing recombinant antibodies against ATL61. Recombinant antibodies have been shown to be "more effective than polyclonal antibodies, and far more reproducible" . While initially more resource-intensive, this approach eliminates batch-to-batch variation for long-term studies.

• Sample archiving strategy: For truly longitudinal studies, process and store critical samples simultaneously, then analyze them together with the same antibody batch. This retrospective approach bypasses batch variation issues entirely.

The financial impact of antibody variability is substantial, with "estimated financial losses of $0.4–1.8 billion per year in the United States alone" due to inadequate antibody characterization. Implementing robust validation protocols for each new batch is therefore not just scientifically sound but economically justified.

How can ATL61 antibody be integrated into multi-omics plant research approaches?

Integrating ATL61 antibody-based methods into multi-omics research frameworks enables comprehensive understanding of RING-H2 finger protein functions in plant biology:

• Antibody-based proteomics with transcriptomics:

  • Use ATL61 antibody for protein quantification via Western blot or ELISA alongside RNA-seq

  • Correlate protein and transcript levels to identify post-transcriptional regulation

  • Investigate discrepancies between protein and mRNA levels to reveal regulatory mechanisms

• Spatial proteomics integration:

  • Employ immunohistochemistry with ATL61 antibody to map tissue-specific expression

  • Combine with single-cell transcriptomics data to create high-resolution spatial-expression maps

  • Correlate localization patterns with tissue-specific metabolomic profiles

• Interactome mapping workflow:

• Phenomics correlation:

  • Track ATL61 protein levels across developmental stages or stress conditions

  • Correlate with phenotypic data from high-throughput phenotyping platforms

  • Develop predictive models linking protein levels to specific plant traits

• Functional validation:

  • Use antibody-mediated protein depletion in conjunction with metabolomic profiling

  • Identify metabolic pathways affected by ATL61 activity

  • Create integrated models of ATL61's role in plant metabolism and development

This multi-omics approach addresses the concern that "characterization data are potentially cell or tissue type specific" by providing multiple layers of evidence across different biological contexts, strengthening the validity and broader applicability of research findings.