ATL49 Antibody

What is ATL49 protein and why is it significant for antibody research?

ATL49 is a putative RING-H2 finger protein primarily found in Helianthus annuus (common sunflower). RING-H2 finger proteins typically function as E3 ubiquitin ligases in the ubiquitin-proteasome pathway, playing crucial roles in protein degradation and cellular regulation. The significance of developing antibodies against ATL49 lies in its potential role in plant stress responses and developmental processes. Methodologically, researchers studying ATL49 should first confirm protein expression patterns across different tissues and environmental conditions to establish a baseline for antibody validation .

What are the recommended expression systems for generating recombinant ATL49 protein for antibody production?

For ATL49 protein expression, E. coli-based systems represent a primary choice due to their high yield and cost-effectiveness. The LOC110865546 gene can be cloned into expression vectors such as pcDNA3.1+/C-(K)DYK, which has been used successfully with this gene. When expressing plant proteins like ATL49 in bacterial systems, considerations should include:

The choice should be guided by the intended application of the antibody and whether conformational epitopes are critical for recognition .

What epitope selection strategies are most effective for ATL49 antibody development?

When developing antibodies against ATL49, epitope selection should focus on unique regions that distinguish it from other RING-H2 finger proteins. Methodologically:

• Perform sequence alignment of ATL49 with homologous proteins to identify unique regions

• Use epitope prediction algorithms to assess antigenicity, hydrophilicity, and surface probability

• Consider the RING-H2 domain structure while avoiding highly conserved zinc-binding motifs

• Target regions outside the RING domain for greater specificity

Linear peptide epitopes (10-20 amino acids) from unique regions of ATL49 often yield more specific antibodies compared to using the full protein, which might generate antibodies cross-reactive with other RING finger proteins. For conformational epitopes, structural information from related RING-H2 proteins should guide epitope design .

How can cross-reactivity with other RING-H2 finger proteins be minimized when developing ATL49-specific antibodies?

Minimizing cross-reactivity requires a multi-faceted approach:

• Bioinformatic analysis: Compare ATL49 sequence with other RING-H2 family members across species, particularly focusing on regions outside the highly conserved RING domain.

• Absorption protocols: Implement pre-absorption of antibody preparations with recombinant proteins of closely related family members.

• Validation methodology:

  • Perform Western blots on tissue samples from wildtype and ATL49-knockout/knockdown lines

  • Test antibody specificity against a panel of recombinant RING-H2 proteins

  • Employ competition assays with predicted cross-reactive epitopes

• Epitope engineering: Consider designing chimeric peptides that incorporate unique ATL49 sequences while minimizing conserved regions.

This comprehensive approach significantly reduces false positives in experimental applications, especially when working with complex plant extracts containing multiple RING-domain proteins .

What are the optimal validation protocols for confirming ATL49 antibody specificity and sensitivity?

A rigorous validation workflow for ATL49 antibodies should include:

For ATL49 specifically, validation should include comparison of expression patterns across plant tissues and under conditions known to regulate RING-H2 proteins (stress responses, hormone treatments). Sensitivity thresholds should be determined using dilution series of recombinant protein, with acceptable detection limits typically in the low nanogram range .

What experimental design considerations are critical when using ATL49 antibodies to study protein-protein interactions?

When investigating ATL49 protein interactions using antibody-based approaches, consider these methodological elements:

• Binding conditions optimization:

  • Test multiple buffer compositions, varying salt concentrations (150-500mM), pH ranges (6.8-8.0), and detergents (0.1-1% NP-40 or Triton X-100)

  • Include protease and deubiquitinase inhibitors to preserve interaction integrity

• Experimental controls:

  • Include non-specific IgG controls from the same species as the ATL49 antibody

  • Implement substrate-trap mutations (e.g., RING domain mutations) as positive controls

  • Use pre-treatment with denaturation conditions as negative controls

• Validation approaches:

  • Confirm interactions using reciprocal immunoprecipitation

  • Employ orthogonal methods such as proximity ligation assays

  • Consider applying FRET or BRET when studying dynamic interactions

• Subcellular considerations:

  • Account for compartmentalization when designing extraction protocols

  • Use cellular fractionation to enrich for expected interaction compartments

These design elements are particularly important for RING-H2 proteins like ATL49, which often form transient interactions with substrate proteins in the ubiquitination pathway .

What techniques are most effective for studying ATL49 post-translational modifications using antibodies?

For studying post-translational modifications (PTMs) of ATL49, particularly auto-ubiquitination common to RING-H2 proteins, consider these methodological approaches:

• Modification-specific antibodies:

  • Develop antibodies against predicted ubiquitination sites on ATL49

  • Utilize phosphorylation-specific antibodies if kinase interaction is suspected

• Sequential immunoprecipitation protocol:

  1. First immunoprecipitation with anti-ATL49 antibody

  2. Elution under mild conditions

  3. Second immunoprecipitation with anti-ubiquitin or anti-phospho antibodies

  4. Analysis by Western blot or mass spectrometry

• In vitro modification assays:

  • Purify recombinant ATL49 using the antibody

  • Perform in vitro ubiquitination assays with E1/E2 enzymes

  • Detect auto-ubiquitination by size shift and ubiquitin-specific antibodies

• Mass spectrometry workflow:

  • Immunoprecipitate ATL49 under denaturing conditions

  • Perform tryptic digestion

  • Analyze by LC-MS/MS focusing on ubiquitination and phosphorylation sites

These techniques should include appropriate controls and consider the potentially transient nature of some modifications on RING-H2 proteins like ATL49 .

How can ATL49 antibodies be effectively used in plant stress response studies?

To effectively employ ATL49 antibodies in plant stress studies:

• Time-course experimental design:

  • Sample collection at multiple timepoints (0, 1, 3, 6, 12, 24, 48 hours) after stress induction

  • Parallel protein and mRNA sampling to correlate transcriptional and translational changes

  • Include recovery phase measurements to assess reversibility

• Stress-specific protocols:

  • For abiotic stresses: Apply controlled drought, salt, heat, or cold treatments

  • For biotic stresses: Use pathogen infiltration or damage-associated molecular patterns

• Tissue-specific analysis:

  • Compare ATL49 expression across different plant tissues under stress

  • Employ immunohistochemistry with the antibody to map spatial regulation

• Functional readouts:

  • Correlate ATL49 protein levels with ubiquitination activity

  • Identify stress-responsive substrates using co-immunoprecipitation

  • Monitor protein stability under different stress conditions

This methodological framework accounts for the dynamic nature of RING-H2 proteins in stress signaling cascades and provides a comprehensive view of ATL49's role in plant stress responses .

What are the recommended approaches for developing multiplex assays that include ATL49 antibodies?

Developing multiplex assays incorporating ATL49 antibodies requires:

• Antibody compatibility assessment:

  • Test for cross-reactivity between primary antibodies

  • Validate specificity when used in combination

  • Ensure secondary antibody compatibility

• Fluorescence-based multiplexing:

  • Select fluorophores with minimal spectral overlap

  • Implement sequential staining for closely related targets

  • Use zenon labeling for antibodies from the same species

• Bead-based multiplex platforms:

  • Conjugate ATL49 antibody to distinctly coded microbeads

  • Optimize antibody concentration for consistent signal-to-noise ratio

  • Develop standard curves using recombinant ATL49 protein

• Spatial multiplexing considerations:

  • For tissue analysis, implement cyclic immunofluorescence with ATL49 antibody

  • Use spectral unmixing algorithms to separate overlapping signals

  • Consider tyramide signal amplification for low-abundance detection

When specifically multiplexing ATL49 with other plant proteins, account for the potentially variable expression levels by balancing antibody concentrations and detection sensitivities across targets .

How should researchers address inconsistent ATL49 antibody performance across different experimental conditions?

When encountering variable ATL49 antibody performance:

• Sample preparation assessment:

  • Evaluate extraction buffer compatibility with antibody epitope recognition

  • Test multiple fixation protocols for imaging applications

  • Consider native vs. denaturing conditions based on epitope accessibility

• Systematic optimization approach:

  • Create a matrix of conditions varying antibody concentration, incubation time, and temperature

  • Test multiple blocking agents (BSA, milk, commercial blockers)

  • Evaluate signal enhancement methods for low-abundance detection

• Batch-to-batch variability management:

  • Maintain reference samples for standardization across experiments

  • Implement internal controls in each experiment

  • Consider generating monoclonal antibodies for critical applications

• Application-specific troubleshooting:

  • For Western blots: Adjust transfer conditions for membrane-bound RING proteins

  • For IHC/IF: Optimize antigen retrieval methods for plant tissues

  • For IP: Test multiple bead types and binding/washing conditions

This systematic approach helps isolate variables affecting antibody performance and establishes reliable protocols for consistent results across experimental conditions .

What are the latest methods for applying AI and computational approaches to enhance ATL49 antibody design and epitope selection?

Recent advances in AI-based antibody design applicable to ATL49 include:

• Zero-shot antibody design frameworks:

  • Generative AI models trained on antibody-antigen interactions

  • Structure-based prediction of binding affinity to ATL49 epitopes

  • In silico screening of millions of potential antibody sequences

• Epitope mapping optimization:

  • Computational analysis of ATL49 structure to identify surface-exposed regions

  • Prediction of B-cell epitopes with machine learning algorithms

  • Molecular dynamics simulations to account for protein flexibility

• High-throughput screening integration:

  • AI-guided library design for phage display or yeast display

  • Automated analysis of binding data to identify successful candidates

  • Iterative optimization through feedback loops

• Developability assessment:

  • Prediction of antibody stability, solubility, and immunogenicity

  • Computational assessment of cross-reactivity risks

  • Virtual affinity maturation to enhance binding properties

These computational approaches significantly reduce experimental timelines and resources required for developing effective ATL49 antibodies by narrowing the design space to the most promising candidates .

How can researchers effectively utilize ATL49 antibodies in single-cell analysis of plant tissues?

For single-cell applications with ATL49 antibodies:

• Tissue preparation protocols:

  • Optimize gentle cell wall digestion using enzyme combinations

  • Implement nuclei isolation protocols for fixed tissues

  • Consider cryosectioning approaches to preserve cellular architecture

• Single-cell protein analysis methods:

  • Adapt CyTOF (mass cytometry) protocols for plant cells using metal-labeled ATL49 antibodies

  • Implement microfluidic antibody capture for quantitative analysis

  • Explore proximity extension assays for sensitive detection

• Spatial transcriptomics integration:

  • Combine ATL49 antibody staining with in situ RNA detection

  • Implement multiplexed epitope and transcript detection

  • Correlate protein levels with transcriptional states

• Data analysis considerations:

  • Develop clustering algorithms specific to plant cell types

  • Implement trajectory analysis to identify developmental patterns

  • Account for autofluorescence common in plant tissues

This methodological framework enables researchers to study cell-type-specific expression and regulation of ATL49, providing insights into its role in plant development and stress responses at single-cell resolution .

What emerging technologies are likely to enhance ATL49 antibody applications in the next 5 years?

Emerging technologies poised to transform ATL49 antibody applications include:

• Nanobody and single-domain antibody alternatives:

  • Development of plant-specific nanobody libraries

  • Enhanced penetration into plant tissues and subcellular compartments

  • Simplified recombinant production systems

• CRISPR-based tagging for validation:

  • Precise endogenous tagging of ATL49 for antibody validation

  • Generation of epitope-tagged knock-in lines in model plants

  • Comparison of tagged protein localization with antibody staining

• Advanced imaging modalities:

  • Super-resolution microscopy protocols optimized for plant tissues

  • Expansion microscopy to visualize subcellular localization

  • Correlative light and electron microscopy with immunogold labeling

• Synthetic biology approaches:

  • Antibody-based biosensors for monitoring ATL49 activity in vivo

  • Split-protein complementation systems for detecting interactions

  • Optogenetic tools combined with antibody-based detection