ATL48 Antibody

What is the ATL48 antibody and what is its target antigen in plant research?

ATL48 antibody is a rabbit polyclonal antibody that specifically targets the RING-H2 finger protein ATL48, which belongs to the ATL (Arabidopsis Toxicos en Levadura) family of E3 ubiquitin ligases found in various plant species . This antibody has been raised against recombinant Arabidopsis thaliana ATL48 protein and is primarily used for detecting ATL48 proteins in plant samples . The target protein (ATL48) contains a RING-H2 finger domain that is essential for E3 ubiquitin ligase activity in plants, making it an important component of protein degradation pathways via the ubiquitin-proteasome system .

The gene encoding this protein has been identified in multiple plant species:

• In Arabidopsis thaliana (Gene Symbol: AT3G48030, UniProt Number: Q7X843)

• In Nicotiana tomentosiformis as "RING-H2 finger protein ATL48-like" (Gene ID: 104089345)

• In Oryza sativa Japonica Group as "RING-H2 finger protein ATL48" (Gene ID: 4346912, Synonym: ATL3I)

What are the validated applications for ATL48 antibody in experimental procedures?

Based on available research data, the ATL48 antibody has been validated for the following applications:

When using ATL48 antibody in Western blotting, researchers should expect to detect the RING-H2 finger protein at its predicted molecular weight. The antibody format (unconjugated, IgG isotype, affinity purified) makes it suitable for these standard immunological techniques when working with plant material .

What is the recommended storage and handling protocol for ATL48 antibody?

For optimal preservation of antibody activity:

• Upon receipt, store ATL48 antibody at -20°C or -80°C to prevent degradation .

• Avoid repeated freeze-thaw cycles, which can cause antibody denaturation and reduced activity.

• The antibody is provided in a buffer containing 0.03% Proclin 300 as a preservative, with 50% Glycerol and 0.01M PBS (pH 7.4) as stabilizers .

• When working with the antibody, aliquot into smaller volumes to minimize freeze-thaw cycles.

• Consider using antibody stabilization approaches (similar to SPEAR technology) for extended use at room temperature if required for plant tissue penetration .

How can I optimize ATL48 antibody performance for detecting low-abundance targets in plant samples?

For detecting low-abundance ATL48 protein in plant samples, implement a comprehensive optimization strategy:

• Signal amplification methods:

  • Utilize tyramide signal amplification (TSA) which can increase sensitivity 10-100 fold

  • Consider biotin-streptavidin amplification systems as secondary detection

• Sample preparation optimization:

  • Enrich target proteins through immunoprecipitation before Western blotting

  • Use plant-specific protein extraction buffers containing appropriate protease inhibitors

  • Consider subcellular fractionation if ATL48 is compartmentalized

• Detection system enhancement:

  • Use high-sensitivity chemiluminescent substrates for Western blotting

  • Increase antibody incubation time (overnight at 4°C) to improve binding kinetics

  • Optimize blocking solutions to reduce background while maintaining specific signal

• Thermal facilitation:

  • Consider adapting the thermally facilitated immunolabeling strategy (ThICK staining) by stabilizing the ATL48 antibody

  • This approach can increase tissue penetration and reduce required antibody concentration

Validation experiment: Compare standard protocols with optimized conditions using serial dilutions of recombinant ATL48 protein to establish a quantitative improvement in detection limit.

What experimental controls are essential when using ATL48 antibody in plant research?

Implementing proper controls is crucial for valid interpretation of results with ATL48 antibody:

Additionally, when performing functional studies on ATL48 (an E3 ubiquitin ligase), include controls for proteasome inhibition to confirm the protein's function in the ubiquitination pathway.

How does sample preparation affect ATL48 antibody performance across different plant tissue types?

Sample preparation significantly impacts ATL48 antibody performance due to the unique challenges of plant tissues:

• Cell wall considerations:

  • Plant cell walls can impede antibody penetration

  • Enzymatic digestion with cellulase/pectinase may improve accessibility

  • For fixed tissues, consider extended permeabilization times

• Protein extraction protocols by tissue type:

  • Leaf tissue: Use buffers containing 1-2% Triton X-100 and PVP to remove phenolic compounds

  • Root tissue: Include higher concentrations of protease inhibitors due to higher protease activity

  • Seed tissue: More aggressive extraction methods needed (e.g., bead beating with SDS buffer)

• Fixation impact:

  • Paraformaldehyde fixation can mask epitopes - optimize concentration and timing

  • Consider antigen retrieval methods like heat-induced epitope retrieval adapted for plant tissues

  • For certain applications, acetone fixation may better preserve ATL48 antigenicity

• Plant-specific interfering compounds:

  • Implement steps to remove plant-specific secondary metabolites that can interfere with antibody binding

  • Consider adding PVP, PVPP, or activated charcoal to binding buffers to adsorb phenolic compounds

  • Include higher BSA concentrations (3-5%) in blocking solutions to reduce non-specific binding

The composition of extraction buffers should be adjusted based on the plant species being studied, as the expression levels and cellular localization of ATL48 may vary significantly across species, impacting detection efficiency.

What are common causes of inconsistent results when using ATL48 antibody, and how can they be resolved?

When encountering variability with ATL48 antibody, systematically address these common issues:

• Inconsistent Western blot signals:

  • Problem: Variable band intensity between replicates

  • Solution: Standardize protein extraction protocol; ensure complete denaturation; implement precise quantification methods; consider using automated Western blot systems for consistent processing

• High background in immunoassays:

  • Problem: Non-specific binding obscuring specific signal

  • Solution: Optimize blocking (try 5% non-fat milk vs. BSA); increase washing stringency; titrate antibody concentration; consider pre-adsorption with plant lysate lacking target

• Cross-reactivity with unintended targets:

  • Problem: Multiple bands or signals in unexpected locations

  • Solution: Increase washing stringency; reduce antibody concentration; validate with knockout controls; consider using peptide competition assays

• Loss of antibody activity:

  • Problem: Diminishing signal over time

  • Solution: Proper storage at -80°C; aliquot to avoid freeze-thaw cycles; implement antibody stabilization methods as described in recent research on thermal stabilization of antibodies

• Inconsistent results between techniques:

  • Problem: Antibody works in ELISA but not Western blot

  • Solution: Different techniques expose different epitopes; optimize denaturation conditions; consider native vs. reducing conditions

Methodical troubleshooting approach:

• Validate antibody activity with positive control (recombinant protein)

• Test pre-immune serum to assess background

• Systematically alter one variable at a time

• Document all conditions precisely for reproducibility

How can I quantitatively analyze and validate ATL48 binding affinity and specificity?

For rigorous quantitative analysis of ATL48 antibody performance:

• Binding affinity determination:

  • Implement biolayer interferometry (BLI) techniques similar to those described in antibody-antigen interaction studies

  • Create a dilution series of antibody against immobilized antigen to generate binding curves

  • Calculate KD values to quantify binding strength using Langmuir binding models

• Specificity validation methods:

  • Perform competitive ELISA with related plant RING-H2 proteins

  • Use Western blot analysis against recombinant ATL48 versus other related ATL family proteins

  • Consider epitope mapping to identify the specific binding region

• Cross-reactivity assessment across species:

  • Test against protein extracts from multiple plant species (Arabidopsis, tobacco, rice)

  • Quantify relative binding affinity across different plant ATL48 homologs

  • Create a cross-reactivity matrix showing affinity values across species

• Advanced validation using surface plasmon resonance:

  • Immobilize recombinant ATL48 on SPR chip

  • Measure association and dissociation rates (kon, koff)

  • Generate comprehensive binding kinetics data

Sample data presentation format:

How can ATL48 antibody be applied in studying plant stress responses and protein degradation pathways?

The ATL48 antibody enables advanced studies of plant stress responses through the following approaches:

• Monitoring ATL48 protein dynamics during stress:

  • Track changes in ATL48 protein levels in response to biotic and abiotic stresses

  • Combine with transcriptomic data to correlate protein and mRNA levels

  • Use time-course experiments to establish temporal regulation patterns

• Ubiquitination pathway analysis:

  • Use ATL48 antibody in co-immunoprecipitation to identify substrate proteins targeted for degradation

  • Combine with ubiquitin antibodies to visualize polyubiquitinated substrates

  • Implement assays to monitor E3 ligase activity in vitro

• Subcellular localization studies:

  • Employ immunofluorescence microscopy to track ATL48 redistribution under stress conditions

  • Use biochemical fractionation followed by Western blotting to quantify compartment-specific changes

  • Correlate localization with functional activity

• Protein interaction network mapping:

  • Utilize ATL48 antibody for proximity-dependent labeling techniques

  • Perform co-immunoprecipitation followed by mass spectrometry to identify interacting partners

  • Validate interactions using reverse co-IP and functional assays

Methodological approach for stress studies:

• Expose plants to multiple stress conditions (drought, salt, pathogen, heat)

• Harvest tissues at multiple time points

• Perform protein extraction and quantification

• Use ATL48 antibody to track protein level changes via Western blot

• Normalize to appropriate housekeeping proteins

• Compare ATL48 dynamics across stress conditions

What emerging technologies can be integrated with ATL48 antibody for advanced plant protein research?

Researchers can leverage cutting-edge technologies with ATL48 antibody:

• Antibody engineering approaches:

  • Consider applying computational antibody design methods to enhance ATL48 antibody specificity

  • Explore direct energy-based preference optimization techniques for improved binding properties

  • Implement rational design principles to optimize ATL48 antibody for specific applications

• Single-cell techniques:

  • Adapt recently developed single-cell antibody-based detection methods for plant cells

  • Implement proximity ligation assays to visualize protein-protein interactions at single-cell resolution

  • Consider microfluidic approaches for high-throughput analysis of ATL48 dynamics

• Automated high-content screening:

  • Develop automated immunofluorescence workflows using ATL48 antibody

  • Implement machine learning for image analysis to detect subtle phenotypic changes

  • Establish quantitative phenotypic profiling methods

• Integration with genetic libraries:

  • Apply ATL48 antibody screening against plant mutant collections

  • Combine with CRISPR screening data to correlate genotype with ATL48-dependent phenotypes

  • Develop active learning approaches similar to those in antibody-antigen binding prediction

• Thermal stabilization for enhanced tissue penetration:

  • Implement antibody stabilization methods as described in recent research

  • Apply thermally accelerated immunostaining to achieve deeper tissue penetration

  • Optimize protocols specifically for plant tissue architecture

Future research potential includes developing ATL48 antibody variants with enhanced specificities for different plant species, enabling comparative studies across evolutionary lineages.

How can database resources be utilized to contextualize ATL48 antibody research findings?

Effectively leverage database resources to enhance ATL48 antibody research:

• Integration with antibody databases:

  • Cross-reference ATL48 binding properties with the Patent and Literature Antibody Database (PLAbDab)

  • Utilize standardized reporting formats for antibody characterization data

  • Contribute validated ATL48 antibody data to community resources

• Structural analysis approaches:

  • Use the Antigen-Antibody Complex Database (AACDB) to model potential binding interfaces

  • Apply information from the database's 7,498 antigen-antibody complexes to predict binding mechanisms

  • Implement computational modeling to predict ATL48-antibody interactions

• Comparative genomics for cross-species research:

  • Utilize genomic databases to identify ATL48 homologs across plant species

  • Predict cross-reactivity based on sequence conservation

  • Design experiments targeting conserved vs. variable regions

• Integration with plant-specific databases:

  • Connect ATL48 function with plant stress databases

  • Correlate ATL48 dynamics with transcriptomic data from public repositories

  • Utilize pathway databases to contextualize ATL48 within the ubiquitin-proteasome system

Data integration workflow:

• Extract ATL48 sequence from UniProt (Q7X843)

• Perform homology searches across plant species

• Cross-reference with functional annotations

• Predict antibody binding sites and accessibility

• Design experiments targeting specific functional domains

By leveraging these database resources, researchers can design more targeted experiments and better interpret results within the broader context of plant biology and antibody-antigen interactions.