ATL59 Antibody

What is ATL59 Antibody and what target does it recognize?

ATL59 Antibody (product code CSB-PA879795XA01DOA) is a research antibody designed for detection of ATL59 protein from Arabidopsis thaliana, also known as mouse-ear cress. According to database information, this antibody targets a protein with UniProt accession number Q9SN27 . ATL59 belongs to the Arabidopsis Tóxicos en Levadura (ATL) family, which typically consists of E3 ubiquitin ligases containing a RING-H2 finger domain and a transmembrane domain. These proteins are involved in various cellular processes including plant defense responses, hormone signaling, and developmental regulation.

What experimental applications are suitable for ATL59 Antibody?

While specific validated applications for ATL59 Antibody are not explicitly detailed in current literature, research-grade antibodies for plant proteins are typically validated for applications including Western blotting, immunoprecipitation (IP), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Similar to approaches used with other research antibodies like AP-2 gamma antibody, experimental conditions should be optimized for each specific application as noted in comparable antibody documentation: "Optimal dilutions should be determined by each laboratory for each application" .

What are recommended storage and handling protocols for ATL59 Antibody?

For optimal performance and longevity, plant-specific antibodies like ATL59 Antibody should generally be stored according to manufacturer specifications. Based on standard practices for research antibodies, this typically includes storage at -20°C for long-term preservation or at 4°C for short-term use. Aliquoting the antibody upon receipt can help prevent protein degradation from repeated freeze-thaw cycles. Similar antibody products are typically available in two formats: lyophilized or as a 0.2 μm filtered solution in phosphate-buffered saline (PBS) .

How should I determine optimal working dilutions for ATL59 Antibody?

Determining the optimal working dilution of ATL59 Antibody requires empirical testing through dilution series experiments for each specific application. For Western blotting applications, based on protocols used with comparable research antibodies, begin testing with dilutions in the range of 1:500 to 1:5000. For immunohistochemistry applications, starting dilutions between 1:50 and 1:500 are typically appropriate. As demonstrated in protocols for other plant antibodies, antibody concentration significantly impacts detection sensitivity and specificity. For example, comparable antibodies are often used at concentrations around 2-10 μg/mL for Western blotting and 10 μg/mL for immunohistochemistry applications .

What controls are essential when performing experiments with ATL59 Antibody?

When designing experiments with ATL59 Antibody, several controls should be incorporated to ensure reliable and interpretable results:

• Positive control: Wild-type Arabidopsis thaliana tissue samples expressing ATL59

• Negative control: Arabidopsis mutant lines with ATL59 deletion or knockdown, or tissues where ATL59 is not expressed

• Primary antibody omission control: To establish baseline background signal

• Secondary antibody-only control: To detect non-specific binding of the secondary detection system

• Loading control: For Western blot applications, detection of housekeeping proteins like actin or GAPDH to normalize protein loading

Similar to protocols established for other antibodies, these controls help distinguish specific signal from experimental artifacts .

What protein extraction methods are recommended for ATL59 detection in plant tissues?

For optimal extraction of ATL59 protein from Arabidopsis tissues, consider these methodological approaches:

• Homogenization in liquid nitrogen followed by extraction in buffer containing:

  • Detergent (e.g., 1% Triton X-100 or NP-40)

  • Protease inhibitor cocktail to prevent degradation

  • Reducing agents (DTT or β-mercaptoethanol)

  • Buffer conditions maintaining pH 7.4-8.0

• For membrane-associated proteins like ATL family members, consider:

  • Addition of stronger detergents (SDS) for complete solubilization

  • Subcellular fractionation to enrich for membrane compartments

  • Modified extraction protocols optimized for RING-finger containing proteins

When analyzing ATL59 by Western blot, reducing conditions are likely necessary, similar to those described for other plant protein detection systems .

What factors might cause weak or absent signal when using ATL59 Antibody?

Several technical and biological factors can contribute to weak or absent signals in ATL59 Antibody experiments:

• Insufficient protein extraction: Plant tissues contain cell walls and secondary metabolites that can interfere with protein extraction efficiency

• Protein denaturation: Improper sample preparation may destroy the epitope recognized by the antibody

• Suboptimal antibody concentration: Too dilute primary or secondary antibody concentrations

• Insufficient antigen retrieval: For fixed tissues, inadequate unmasking of epitopes

• Developmental or tissue-specific expression: ATL59 may be expressed only under specific conditions or in certain tissues

• Post-translational modifications: Modifications may mask the epitope recognized by the antibody

For optimal detection, conditions should be systematically optimized similar to approaches used for other plant antibodies .

How can background signal be reduced when using ATL59 Antibody?

High background signal can compromise experimental interpretation. To reduce non-specific binding and background:

• Optimize blocking conditions: Increase blocking agent concentration (BSA, non-fat milk, or plant-specific blockers) and extend blocking time

• Adjust antibody concentration: Dilute primary and secondary antibodies appropriately

• Enhance washing steps: Increase number and duration of washes between incubation steps

• Add detergents: Include 0.05-0.1% Tween-20 in wash buffers

• Pre-absorb antibody: Incubate with non-specific proteins before application to samples

• Use plant-specific blocking agents: Consider adding non-specific plant extracts to reduce binding to common plant components

Similar approaches have been demonstrated effective in reducing background when using other plant-specific antibodies .

How can I validate the specificity of ATL59 Antibody detection?

Rigorous validation of antibody specificity is crucial for reliable research findings. For ATL59 Antibody, consider:

• Genetic validation: Test antibody on atl59 knockout or knockdown lines

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

• Molecular weight verification: Confirm detected bands match predicted molecular weight of ATL59

• Multiple detection methods: Compare results across different techniques (Western blot, immunohistochemistry)

• Correlation with transcript data: Compare protein detection with known ATL59 mRNA expression patterns

Such validation approaches align with best practices established for research antibodies in plant biology .

How can ATL59 Antibody be used to study protein-protein interactions?

ATL59 Antibody can be employed to investigate protein interaction networks using these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare non-denaturing lysates from Arabidopsis tissues

  • Immunoprecipitate using ATL59 Antibody

  • Identify co-precipitated proteins via mass spectrometry or Western blotting

• Proximity-dependent labeling:

  • Combine with BioID or APEX2 approaches for in vivo interaction studies

  • Use ATL59 Antibody to validate proximity labeling results

• Immunofluorescence co-localization:

  • Perform dual-labeling with ATL59 Antibody and antibodies against potential interactors

  • Analyze co-localization using confocal microscopy

These approaches can help elucidate the functional roles of ATL59 in ubiquitin-mediated signaling pathways, similar to methodologies used to study other regulatory proteins .

What approaches can be used to study ATL59 in stress response pathways?

Many ATL family proteins are implicated in stress responses. To investigate ATL59's role:

• Stress induction experiments:

  • Subject plants to various stressors (drought, pathogens, hormones)

  • Use ATL59 Antibody to monitor protein level changes via Western blot

  • Quantify relative expression compared to control conditions

• Subcellular localization studies:

  • Perform fractionation followed by Western blotting with ATL59 Antibody

  • Monitor potential relocalization during stress response

  • Compare with known stress response markers

• Post-translational modification analysis:

  • Use ATL59 Antibody to immunoprecipitate the protein under different conditions

  • Analyze modifications (phosphorylation, ubiquitination) by mass spectrometry

These approaches can provide insights into ATL59's role in stress signaling networks, similar to methodologies used to study other regulatory proteins in signaling cascades .

How can ATL59 Antibody be used in combination with genetic approaches?

Integrating antibody-based detection with genetic manipulation provides powerful insights:

• Complementation studies:

  • Express modified versions of ATL59 in knockout backgrounds

  • Use ATL59 Antibody to verify expression levels

  • Correlate protein expression with phenotypic rescue

• Protein-domain function analysis:

  • Generate plants expressing truncated or mutated ATL59 versions

  • Use the antibody to confirm expression and stability

  • Map functional domains through correlation with phenotypic effects

• Tissue-specific or inducible expression:

  • Create lines with modified ATL59 expression patterns

  • Use the antibody to validate spatial and temporal expression

  • Correlate with developmental or stress response phenotypes

This combined genetic-immunological approach can reveal functional mechanisms, similar to methodologies used to study regulatory proteins in other systems .

How can I use ATL59 Antibody to compare expression across different Arabidopsis ecotypes?

Natural variation studies using ATL59 Antibody can reveal evolutionary adaptations:

Analysis should include:

• Standardized protein extraction protocols across ecotypes

• Normalized loading using consistent housekeeping proteins

• Statistical analysis of biological replicates (minimum n=3)

• Correlation with any known phenotypic differences between ecotypes

This approach enables identification of natural variation in ATL59 expression that may correlate with adaptive traits .

What are the considerations when comparing ATL family proteins in Arabidopsis?

The ATL family in Arabidopsis comprises numerous members with potentially overlapping functions. When comparing family members:

• Epitope specificity:

  • Determine if ATL59 Antibody might cross-react with closely related ATL proteins

  • Perform sequence alignment of epitope regions to predict potential cross-reactivity

  • Validate specificity using knockout lines for multiple ATL members

• Expression pattern comparison:

  • Use specific antibodies for different ATL members in parallel experiments

  • Create expression tables comparing tissue distribution

  • Correlate with publicly available transcriptome data

• Functional redundancy assessment:

  • Study protein levels in single and multiple atl mutant lines

  • Look for compensatory expression changes among family members

This comparative approach can reveal functional specialization or redundancy within the ATL family, similar to approaches used in studying other protein families .

How does ATL59 protein abundance correlate with transcript levels?

Post-transcriptional regulation can cause discrepancies between mRNA and protein levels:

• Integrated analysis approach:

  • Perform parallel qRT-PCR and Western blot with ATL59 Antibody

  • Calculate correlation coefficients between transcript and protein

  • Analyze samples across developmental stages and stress conditions

• Potential regulatory mechanisms to investigate:

  • miRNA-mediated regulation

  • Protein stability differences

  • Translational efficiency variation

• Time-course analysis:

  • Monitor both transcript and protein levels following stimuli

  • Quantify temporal relationships between mRNA and protein changes

This integrated approach can reveal regulatory mechanisms controlling ATL59 expression, similar to methodologies used to study other plant regulatory proteins .

How might ATL59 Antibody contribute to understanding plant ubiquitin-mediated signaling?

As an E3 ubiquitin ligase family member, ATL59 likely participates in protein degradation pathways:

• Target identification:

  • Use ATL59 Antibody for co-immunoprecipitation followed by mass spectrometry

  • Verify interactions with candidate substrates

  • Monitor ubiquitination status of potential targets

• Pathway integration:

  • Investigate ATL59's role in hormonal signaling networks

  • Study relationships with known ubiquitin pathway components

  • Analyze effects of proteasome inhibitors on ATL59 levels and activity

• Spatial regulation:

  • Use immunohistochemistry to map ATL59 distribution across tissues and cell types

  • Correlate with known sites of active ubiquitin-mediated regulation

These approaches align with current research trends investigating protein quality control mechanisms in plants under normal and stress conditions .

What emerging technologies might enhance ATL59 Antibody applications?

Several cutting-edge approaches could extend ATL59 Antibody utility:

• Super-resolution microscopy:

  • Apply advanced imaging techniques for subcellular localization studies

  • Investigate co-localization with interaction partners at nanometer resolution

• Quantitative proteomics integration:

  • Combine ATL59 Antibody immunoprecipitation with multiplexed protein quantification

  • Map dynamic interaction networks under different conditions

• Single-cell analysis:

  • Adapt ATL59 immunodetection for single-cell profiling

  • Investigate cell-specific expression patterns within complex tissues

These technological integrations represent frontier approaches in plant molecular biology research that could significantly advance understanding of ATL59 function .