ATL58 Antibody

What is ATL58 and what is its role in plant biology?

ATL58 (Q570X5) is a protein found in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology research. It belongs to the ATL (Arabidopsis Tóxicos en Levadura) family of proteins, which are RING-H2 type E3 ubiquitin ligases involved in various cellular processes including stress responses and development. The protein contains specific domains that facilitate protein-protein interactions and ubiquitination activities. When studying ATL58, it's important to consider its subcellular localization and potential interaction partners to properly interpret experimental results .

What are the key specifications of the ATL58 Antibody?

The ATL58 Antibody (CSB-PA681984XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana ATL58 protein. Its specificity is limited to Arabidopsis thaliana samples. The antibody is supplied in liquid form, non-conjugated, and purified using antigen affinity purification methods. The storage buffer consists of 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. Being a polyclonal IgG antibody, it recognizes multiple epitopes on the target protein, which can provide enhanced detection sensitivity in various applications .

For which applications has the ATL58 Antibody been validated?

The ATL58 Antibody has been specifically tested and validated for Enzyme-Linked Immunosorbent Assay (ELISA) and Western Blot (WB) applications. When employing this antibody in Western blotting, researchers should expect to detect the ATL58 protein at its predicted molecular weight, though post-translational modifications may affect observed migration patterns. It is important to note that the antibody has not been validated for other applications such as immunohistochemistry, immunofluorescence, or immunoprecipitation. Any use beyond the validated applications would require thorough optimization and validation by the researcher .

What are the recommended storage conditions for ATL58 Antibody?

To maintain optimal activity, the ATL58 Antibody should be stored at either -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be avoided as they can significantly degrade antibody quality and performance. When working with the antibody, it's advisable to aliquot it into smaller volumes before freezing to minimize freeze-thaw cycles. The storage buffer containing 50% glycerol helps stabilize the antibody during freeze-thaw, but proper handling remains essential for long-term preservation of activity .

What controls should be included when working with ATL58 Antibody?

When designing experiments using the ATL58 Antibody, several controls should be incorporated to ensure reliable interpretation of results:

These controls are essential for proper validation and interpretation of experimental results, especially when employing the antibody in novel contexts or with modified protocols .

How can I validate the specificity of ATL58 Antibody in my experimental system?

To validate ATL58 Antibody specificity in your experimental system, apply a multi-pronged approach following the "five pillars" of antibody validation:

• Genetic strategy: Utilize ATL58 knockout or knockdown Arabidopsis lines as negative controls. The absence or reduction of signal in these samples strongly supports antibody specificity. CRISPR-Cas9 or RNAi techniques can generate appropriate negative control samples.

• Orthogonal strategy: Compare ATL58 protein levels detected by the antibody with mRNA levels measured by RT-qPCR. Correlation between protein and transcript levels under various conditions provides evidence for antibody specificity.

• Independent antibody strategy: If available, compare results with a different antibody targeting ATL58 at a distinct epitope. Concordant results between different antibodies increase confidence in specificity.

• Recombinant expression strategy: Overexpress tagged ATL58 and confirm detection by both the antibody and tag-specific detection methods. This approach can also help determine detection limits.

• Immunoprecipitation-MS strategy: Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody. This comprehensive approach identifies potential cross-reactivity issues .

Documentation of these validation steps is critical for publication and ensuring reproducible research. Remember that antibody performance may vary across different experimental conditions, requiring validation for each specific application and context .

What strategies can optimize Western blotting protocols with ATL58 Antibody?

Optimizing Western blotting with ATL58 Antibody requires systematic adjustment of multiple parameters:

• Sample preparation:

  • Extract proteins using buffers containing protease inhibitors to prevent degradation

  • Include reducing agents (e.g., DTT, β-mercaptoethanol) to ensure proper protein denaturation

  • Heat samples at 95°C for 5 minutes for complete denaturation

• Antibody dilution optimization:

  • Begin with manufacturer's recommended dilution (typically 1:1000)

  • Test serial dilutions (e.g., 1:500, 1:1000, 1:2000) to determine optimal signal-to-noise ratio

  • Record precise antibody lot numbers for reproducibility

• Blocking optimization:

  • Test different blocking agents (BSA, non-fat milk, commercial blockers)

  • Determine optimal blocking time and temperature

  • Consider using the same blocking agent in antibody dilution buffer

• Membrane selection:

  • PVDF membranes typically provide better protein retention and sensitivity

  • Optimize transfer conditions for the predicted molecular weight of ATL58

• Signal development:

  • For low abundance targets, consider enhanced chemiluminescence or fluorescent detection systems

  • Optimize exposure times for chemiluminescence detection

Document all optimization steps methodically in a laboratory notebook for reproducibility. Remember that optimization is an iterative process, and conditions may need adjustment for different experimental systems or sample types .

How can I troubleshoot non-specific binding when using ATL58 Antibody?

Non-specific binding is a common challenge when working with polyclonal antibodies. To troubleshoot and minimize this issue with ATL58 Antibody:

• Adjust blocking conditions:

  • Increase blocking time (from 1 hour to overnight)

  • Test different blocking agents (5% BSA often reduces background compared to milk for phospho-specific detection)

  • Add 0.1-0.3% Tween-20 to washing and antibody incubation buffers

• Optimize antibody concentration:

  • Dilute the antibody further if background is high

  • Reduce incubation time or temperature

• Implement additional washing steps:

  • Increase the number and duration of washes

  • Use more stringent washing buffers (higher salt concentration or detergent)

• Pre-absorb the antibody:

  • Incubate with proteins from non-target species or tissues

  • Use lysates from ATL58 knockout plants to pre-absorb non-specific antibodies

• Cross-adsorption technique:

  • Identify the molecular weight of non-specific bands

  • Use knockout verification to confirm which bands are specific

  • If possible, cut membranes to isolate target molecular weight regions

For complex plant samples with high endogenous peroxidase activity, consider hydrogen peroxide pre-treatment or alternative detection methods. Document all troubleshooting steps methodically to establish a reproducible protocol for your specific experimental system .

What methodologies are recommended for quantifying ATL58 expression using this antibody?

For accurate quantification of ATL58 expression, several methodological approaches are recommended:

• Densitometry analysis:

  • Capture images within the linear dynamic range of detection

  • Use analysis software (ImageJ, ImageLab) to quantify band intensity

  • Always normalize to loading controls (actin, tubulin, total protein stain)

  • Include a standard curve with known quantities of recombinant protein

• Quantitative immunoblotting:

  • Use fluorescently-labeled secondary antibodies for more accurate quantification

  • Include a dilution series of control samples to ensure measurements within linear range

  • Employ multiplexing to detect target and loading control simultaneously

• ELISA-based quantification:

  • Develop a sandwich ELISA using ATL58 Antibody as capture or detection antibody

  • Generate a standard curve using recombinant ATL58 protein

  • Ensure sample preparation methods maintain native protein conformation

• Normalization approaches:

  • For accurate quantification across multiple samples, normalize to:

    • Total protein (measured by stain-free technology or Ponceau S)

    • Housekeeping proteins (after validating their stability under your experimental conditions)

    • Multiple reference proteins rather than a single housekeeping gene

Document all quantification parameters, including exposure settings, analysis methods, and normalization strategies, to ensure reproducibility and reliable comparative analyses across experiments .

How does the polyclonal nature of ATL58 Antibody impact experimental design and interpretation?

The polyclonal nature of the ATL58 Antibody has several significant implications for experimental design and data interpretation:

• Epitope recognition diversity:

  • Polyclonal antibodies recognize multiple epitopes on the target protein

  • Advantages: Enhanced sensitivity, better tolerance to minor protein denaturation or modification

  • Limitations: Increased potential for cross-reactivity with similar epitopes on non-target proteins

• Lot-to-lot variability:

  • Different production lots may contain varying antibody compositions

  • Critical consideration: Document lot numbers for reproducibility

  • Recommendation: Purchase sufficient quantity of a single lot for entire project duration

• Post-translational modification detection:

  • Some antibodies in the polyclonal mixture may recognize epitopes affected by PTMs

  • This can result in detecting multiple bands corresponding to different forms of ATL58

  • Verification approach: Use dephosphorylation or deglycosylation treatments to confirm band identity

• Signal interpretation:

  • Higher sensitivity may detect low-abundance forms of the protein not visible with monoclonal antibodies

  • May require additional controls to distinguish specific from non-specific signals

  • Consider complementary approaches for critical findings (e.g., mass spectrometry)

To mitigate these challenges, implement thorough validation using genetic controls (knockout/knockdown lines), include reciprocal confirmation with orthogonal techniques, and maintain consistent experimental conditions across studies. For publications, explicitly report antibody validation methods, lot numbers, and any observed limitations in specificity or reproducibility .

What are the best practices for using ATL58 Antibody in immunofluorescence applications?

While the ATL58 Antibody has not been specifically validated for immunofluorescence applications, researchers may adapt it for this purpose following these best practices:

• Sample preparation optimization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, acetone)

  • Evaluate different permeabilization protocols (0.1-0.5% Triton X-100, saponin)

  • Consider antigen retrieval techniques if working with fixed tissue sections

• Antibody validation for IF:

  • Begin with positive controls (tissues known to express ATL58)

  • Include negative controls (ATL58 knockout tissue or non-plant tissue)

  • Compare staining patterns with published subcellular localization data

• Signal enhancement techniques:

  • Implement tyramide signal amplification for low-abundance targets

  • Use high-sensitivity detection systems (e.g., quantum dots, highly cross-adsorbed secondaries)

  • Optimize signal-to-noise ratio through careful titration of primary and secondary antibodies

• Counterstaining and colocalization:

  • Use well-established organelle markers to determine subcellular localization

  • Employ nuclear counterstains (DAPI, Hoechst) for orientation

  • Apply appropriate controls for spectral bleed-through in multi-color imaging

• Image acquisition and analysis:

  • Capture images using consistent settings across experimental conditions

  • Implement blind analysis where possible to prevent bias

  • Use appropriate colocalization analysis methods and statistics

Since this application extends beyond the manufacturer's validated uses, thorough controls and stepwise optimization are essential. Document the entire protocol development process carefully and consider conducting parallel experiments with orthogonal approaches (e.g., fluorescent protein tagging) to confirm observed localization patterns .

How can I integrate ATL58 Antibody data with other molecular techniques for comprehensive analysis?

Integrating ATL58 Antibody data with complementary molecular techniques provides a more comprehensive understanding of ATL58 biology. Consider these methodological approaches:

• Multi-omics integration:

  • Correlate protein expression data (Western blot/ELISA) with transcriptomics (RNA-seq, qRT-PCR)

  • Compare protein abundance with promoter activity (reporter assays)

  • Integrate with metabolomics data to identify functional pathways affected by ATL58

• Protein-protein interaction studies:

  • Use ATL58 Antibody for co-immunoprecipitation followed by mass spectrometry

  • Validate interactions using reciprocal pulldowns or proximity ligation assays

  • Correlate interaction data with functional assays (e.g., ubiquitination assays)

• Functional genomics correlation:

  • Compare phenotypes from ATL58 mutant/overexpression lines with protein expression patterns

  • Correlate protein expression with physiological or developmental parameters

  • Integrate with ChIP-seq data if studying transcriptional regulation

• Spatio-temporal analysis:

  • Combine tissue-specific Western blot quantification with in situ hybridization

  • Correlate developmental expression patterns with known developmental markers

  • Compare protein stability/turnover (pulse-chase) with functional readouts

• Data integration frameworks:

  • Employ computational approaches to integrate multiple data types

  • Use pathway analysis tools to place ATL58 in functional networks

  • Apply machine learning for pattern recognition across diverse datasets

What considerations are important when using ATL58 Antibody in plant stress response studies?

When employing ATL58 Antibody for plant stress response studies, several methodological considerations are critical:

• Baseline expression profiling:

  • Establish normal ATL58 expression patterns across tissues and developmental stages

  • Document diurnal variations in expression under standard conditions

  • Create a reference dataset before introducing stress variables

• Stress treatment standardization:

  • Precisely define stress parameters (duration, intensity, application method)

  • Include appropriate positive controls (known stress-responsive proteins)

  • Consider time-course experiments to capture dynamic responses

• Sample preparation adaptations:

  • Modify extraction buffers for stress-treated samples (may contain different interfering compounds)

  • Adjust protein extraction protocols for tissues with altered physical properties post-stress

  • Include additional protease inhibitors for tissues with stress-induced proteolytic activity

• Post-translational modification analysis:

  • Assess potential stress-induced modifications of ATL58 (phosphorylation, ubiquitination)

  • Use phosphatase treatments to determine if multiple bands represent phosphorylated forms

  • Consider specialized techniques (Phos-tag gels) for better resolution of modified forms

• Data normalization challenges:

  • Traditional housekeeping genes may vary under stress conditions

  • Implement multiple normalization strategies (total protein, multiple reference proteins)

  • Validate stability of reference genes under your specific stress conditions

For meaningful interpretation, compare ATL58 expression patterns with known stress markers and integrate findings with physiological measurements. Document all environmental parameters during experiments, as minor variations can significantly impact stress responses in plants .

How should I approach epitope mapping with ATL58 Antibody?

Epitope mapping provides valuable insights into the specific regions of ATL58 recognized by the antibody, which can inform experimental design and interpretation. Follow these methodological approaches:

• Peptide array mapping:

  • Synthesize overlapping peptides spanning the entire ATL58 sequence

  • Probe arrays with the ATL58 Antibody

  • Identify peptides generating positive signals to localize epitope regions

• Truncation mutant analysis:

  • Generate recombinant fragments of ATL58 with sequential deletions

  • Perform Western blot analysis to determine which fragments maintain antibody recognition

  • Narrow down the epitope region through systematic deletion mapping

• Point mutation strategy:

  • Once approximate epitope regions are identified, introduce point mutations

  • Analyze changes in antibody binding affinity with mutated proteins

  • Identify critical amino acids essential for antibody recognition

• Competitive binding assays:

  • Synthesize candidate epitope peptides

  • Perform competition experiments where peptides block antibody binding

  • Quantify inhibition to confirm epitope regions

• Cross-species reactivity analysis:

  • Compare ATL58 sequences across related plant species

  • Test antibody against homologs from different species

  • Correlate recognition patterns with sequence conservation

Understanding epitope regions has practical applications for:

• Predicting potential cross-reactivity with related proteins

• Determining if the antibody can detect denatured vs. native protein

• Assessing whether post-translational modifications might affect recognition

• Interpreting negative results when epitopes might be masked by protein interactions

Document epitope information thoroughly to provide context for experimental results and facilitate method development by other researchers .

What are appropriate approaches for using ATL58 Antibody in multiplex analysis systems?

Multiplexing with ATL58 Antibody requires careful methodological considerations to ensure reliable simultaneous detection of multiple targets:

• Antibody compatibility assessment:

  • Verify that ATL58 Antibody can be used alongside other primary antibodies

  • Test for potential cross-reactivity between antibody pairs

  • Ensure primary antibodies are raised in different host species for compatible secondary detection

• Multiplex Western blotting approaches:

  • Fluorescent Western blotting with spectrally distinct secondary antibodies

  • Sequential reprobing with stripping between detections (validate complete stripping)

  • Size-based multiplexing when target proteins have sufficiently different molecular weights

• Multiplex immunoassay optimization:

  • For bead-based multiplex assays, validate ATL58 Antibody performance in multiplex format

  • Test for potential cross-reactivity with other capture or detection antibodies

  • Establish appropriate dilution factors that work across all targets

• Signal separation strategies:

  • Spectral unmixing for fluorescent detection systems

  • Sequential scanning for narrowly separated emission spectra

  • Spatial separation strategies for tissue section analysis

• Data analysis considerations:

  • Implement appropriate normalization for each target in the multiplex panel

  • Account for potential signal spillover in closely related detection channels

  • Apply statistical methods appropriate for multi-parameter data

For all multiplex applications, comprehensive validation with appropriate single-plex controls is essential. Document all optimization steps and validation experiments thoroughly to ensure reproducible results and proper interpretation of complex datasets .