ATL46 Antibody

What is ATL46 in Arabidopsis thaliana?

ATL46 (Uniprot accession: Q9FL07) is a protein found in Arabidopsis thaliana, a small plant in the mustard family that serves as a model system for research in plant biology . It belongs to the ATL (Arabidopsis Tóxicos en Levadura) family of RING-H2 finger proteins that function as E3 ubiquitin ligases, involved in protein degradation pathways and various stress responses in plants. These proteins play critical roles in plant development and response to environmental stimuli.

What are the key specifications of commercially available ATL46 antibodies?

The commercially available ATL46 antibody (e.g., CSB-PA875486XA01DOA) is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana ATL46 protein . It is purified using antigen affinity methods and supplied in liquid form. The storage buffer typically contains 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . The antibody has been validated for applications including ELISA and Western blotting.

What are the recommended storage conditions for ATL46 antibodies?

ATL46 antibodies should be stored at -20°C or -80°C immediately upon receipt . It's crucial to avoid repeated freeze-thaw cycles as these can compromise antibody stability and activity. For working solutions, small aliquots should be prepared and stored separately to minimize freeze-thaw cycles. When handling the antibody, allow it to thaw completely at room temperature before use and mix gently to ensure homogeneity.

What experimental applications are ATL46 antibodies validated for?

ATL46 antibodies are primarily validated for ELISA and Western blot applications . For Western blotting, these antibodies can detect denatured ATL46 protein, making them valuable for studying protein expression levels, molecular weight confirmation, and post-translational modifications. For ELISA, they enable quantitative detection of ATL46 in plant extracts. While not explicitly validated, these antibodies may also be suitable for immunoprecipitation, immunohistochemistry, and flow cytometry, though optimization would be necessary.

What are the recommended working dilutions for ATL46 antibodies?

Optimal working dilutions for ATL46 antibodies vary by application and should be determined empirically for each experimental setup. Typically, initial testing ranges are:

Always include appropriate controls to validate specificity and minimize background signal.

What sample preparation protocols work best for ATL46 detection in plant tissues?

For optimal ATL46 detection in plant tissues, consider the following protocol:

• Harvest fresh Arabidopsis tissue (preferably 100-200 mg) and flash-freeze in liquid nitrogen

• Grind tissue to a fine powder using a mortar and pestle while maintaining frozen state

• Add extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor cocktail

• Homogenize thoroughly and incubate on ice for 30 minutes with occasional vortexing

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and quantify protein concentration using Bradford or BCA assay

• For Western blot, add sample buffer and heat at 95°C for 5 minutes before loading

For membrane-associated proteins like ATL46, inclusion of appropriate detergents is crucial for solubilization and extraction efficiency.

How can I validate the specificity of ATL46 antibodies in my experimental system?

Validating ATL46 antibody specificity is crucial for reliable experimental results. Consider implementing these approaches:

• Genetic controls: Compare wild-type Arabidopsis with atl46 knockout/knockdown lines to confirm absence or reduction of signal in mutant lines

• Blocking peptide experiment: Pre-incubate the antibody with excess immunizing peptide before application to samples; specific signals should be abolished

• Recombinant protein detection: Use purified recombinant ATL46 protein as a positive control to confirm the correct molecular weight band

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm identity of detected proteins

• Signal reduction in RNAi experiments: Demonstrate correlation between reduced ATL46 mRNA levels and reduced protein signal

When multiple validation methods consistently support antibody specificity, confidence in experimental results increases substantially.

What are the common technical issues in Western blotting with ATL46 antibodies and how can they be resolved?

When performing Western blots with ATL46 antibodies, researchers commonly encounter several technical challenges:

For ATL46 specifically, its membrane association may require optimization of extraction conditions to ensure consistent solubilization and detection.

How can I use ATL46 antibodies for co-immunoprecipitation studies?

Co-immunoprecipitation (Co-IP) with ATL46 antibodies can reveal protein-protein interactions relevant to ATL46's biological function. For optimal results:

• Use a gentler lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40) to preserve protein-protein interactions

• Pre-clear lysate with Protein A/G beads to reduce non-specific binding

• Incubate cleared lysate with ATL46 antibody (approximately 2-5 μg per mg of total protein) overnight at 4°C

• Add pre-washed Protein A/G beads and incubate for 1-2 hours at 4°C with gentle rotation

• Wash beads thoroughly (4-5 times) with wash buffer

• Elute bound proteins with sample buffer for SDS-PAGE analysis

• Analyze co-immunoprecipitated proteins by Western blot or mass spectrometry

Include appropriate controls such as IgG from the same species and lysate-only samples. For interactome studies, consider using stable isotope labeling approaches to distinguish true interactors from background proteins.

What are the advantages and limitations of polyclonal versus monoclonal ATL46 antibodies?

Understanding the differences between polyclonal and monoclonal antibodies is crucial for experimental planning:

Currently, polyclonal antibodies are more commonly available for ATL46 . When selecting an antibody, consider your experimental requirements: for exploratory studies detecting ATL46 under various conditions, polyclonal antibodies offer flexibility; for highly specific detection or quantification, monoclonal antibodies may be preferable once available.

How can ATL46 antibodies be used to study protein-protein interactions in ubiquitination pathways?

As ATL46 likely functions as an E3 ubiquitin ligase, investigating its role in ubiquitination pathways is a valuable research direction. Consider these methodological approaches:

• Sequential immunoprecipitation: First immunoprecipitate with anti-ubiquitin antibodies, then probe with ATL46 antibodies (or vice versa) to detect ubiquitinated forms of ATL46 or its substrates

• In vitro ubiquitination assays: Combine purified E1, E2, ATL46 (as E3), and ubiquitin with potential substrates, then detect ubiquitination using Western blotting

• Proximity ligation assay (PLA): Use ATL46 antibodies in combination with antibodies against potential interaction partners to visualize protein-protein interactions in situ

• Bimolecular fluorescence complementation (BiFC): Complement with genetic approaches to confirm interactions detected with antibody-based methods

• Mass spectrometry of ATL46 complexes: Immunoprecipitate with ATL46 antibodies and identify interacting proteins by mass spectrometry

When studying dynamic processes like ubiquitination, include proteasome inhibitors (e.g., MG132) in your experimental design to stabilize otherwise rapidly degraded ubiquitinated proteins.

What are the best practices for quantifying ATL46 expression levels using antibody-based methods?

For accurate quantification of ATL46 expression:

• Western blot quantification:

  • Use housekeeping proteins (e.g., actin, tubulin) as loading controls

  • Ensure signal is within linear range of detection

  • Use image analysis software (ImageJ, Image Lab) for densitometry

  • Normalize ATL46 band intensity to loading control

  • Include a standard curve of recombinant ATL46 for absolute quantification

• ELISA quantification:

  • Develop a standard curve using recombinant ATL46 protein

  • Ensure sample dilutions fall within the linear range of the standard curve

  • Run technical triplicates to assess variability

  • Include negative controls from atl46 knockout plants if available

• Statistical analysis:

  • Apply appropriate statistical tests based on experimental design

  • Report both biological and technical replication

  • Consider normalized relative quantities rather than absolute values when comparing between experiments

For time-course studies or treatments affecting global protein expression, multiple normalization controls should be used to ensure accurate interpretation.

How might ATL46 antibodies be utilized in plant stress response research?

ATL family proteins often play important roles in plant responses to biotic and abiotic stresses. Researchers can leverage ATL46 antibodies to:

• Monitor expression changes: Track ATL46 protein levels during exposure to different stressors (drought, salinity, pathogens)

• Identify tissue-specific responses: Use immunohistochemistry to localize ATL46 expression in different plant tissues under stress conditions

• Characterize post-translational modifications: Detect changes in ATL46 phosphorylation, ubiquitination, or other modifications during stress responses

• Identify stress-specific interaction partners: Perform co-immunoprecipitation under different stress conditions to map condition-specific protein complexes

• Develop biosensors: Create antibody-based detection systems for monitoring plant stress in agricultural settings

These approaches can help elucidate the molecular mechanisms underlying plant adaptation to environmental challenges, potentially informing strategies for improving crop resilience.

What considerations are important when using ATL46 antibodies in different plant species?

When extending ATL46 antibody applications beyond Arabidopsis:

• Sequence homology analysis: Compare ATL46 sequence between Arabidopsis and target species to assess potential cross-reactivity

• Epitope conservation: Determine if the immunogenic region used to generate the antibody is conserved in the target species

• Validation in new species: Always validate antibody reactivity in the new species using positive and negative controls

• Optimization for tissue type: Different plant tissues may require modified extraction protocols for optimal results

• Species-specific controls: Include appropriate controls from the target species rather than relying solely on Arabidopsis controls

A Western blot comparing Arabidopsis and the target species can provide initial evidence of cross-reactivity, which should be followed by more rigorous validation if the antibody will be used extensively in the new species.