ATL18 Antibody

What is ATL18 and what cellular functions does it serve in Arabidopsis thaliana?

ATL18 (AT4G38140) is a protein found in the model plant organism Arabidopsis thaliana. It belongs to the ATL (Arabidopsis Tóxicos en Levadura) family of RING-H2 finger proteins that function as E3 ubiquitin ligases in the ubiquitin-proteasome system. These proteins play crucial roles in protein degradation pathways and are involved in various cellular processes including plant development, hormone signaling, and response to biotic and abiotic stresses. The specific function of ATL18 involves regulating protein turnover through ubiquitination, thereby influencing plant responses to environmental conditions and developmental cues .

What are the primary applications for ATL18 antibody in plant research?

The ATL18 antibody has been validated for Western blot (WB) and ELISA applications in plant research. These techniques allow researchers to detect, quantify, and characterize the ATL18 protein in various experimental contexts. Western blotting provides information about protein expression levels and molecular weight, while ELISA enables quantitative analysis of ATL18 in plant tissue extracts. These applications support investigations into protein expression patterns during different developmental stages, stress responses, and mutant phenotype characterization .

What sample preparation methods are recommended for optimal ATL18 detection in plant tissues?

For optimal detection of ATL18 in plant tissues, sample preparation should involve careful extraction of total proteins while maintaining native protein structure. A recommended protocol includes: 1) Grinding fresh or flash-frozen plant tissue in liquid nitrogen; 2) Extracting proteins in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail; 3) Clearing lysates by centrifugation at 12,000g for 15 minutes at 4°C; 4) Determining protein concentration using Bradford or BCA assay; and 5) Adding appropriate amount of sample buffer and heating at 95°C for 5 minutes before loading for Western blot analysis. For ELISA applications, similar extraction principles apply, though detergent concentrations may need adjustment depending on the specific ELISA protocol employed .

How should researchers design controls when using ATL18 antibody in experimental workflows?

Proper control design is critical for reliable interpretation of ATL18 antibody results. For Western blot applications, recommended controls include: 1) Positive control using the recombinant immunogen protein/peptide provided with the antibody; 2) Negative control using pre-immune serum to assess non-specific binding; 3) Loading control with housekeeping proteins such as actin or tubulin to normalize protein loading; 4) atl18 knockout/knockdown plant material as genetic negative control; and 5) Peptide competition assay to confirm antibody specificity. For ELISA experiments, standard curves using recombinant ATL18 protein, blanks without primary antibody, and samples from knockout plants should be included. These comprehensive controls help distinguish between specific signal and background, ensuring experimental rigor .

What is the recommended antibody dilution range for different applications involving ATL18 antibody?

Based on standard immunological practices for polyclonal antibodies, the recommended dilution ranges for ATL18 antibody are:

Researchers should perform preliminary experiments to determine the optimal dilution for their specific sample type and experimental conditions. Factors affecting optimal dilution include protein expression level, sample preparation method, and detection system sensitivity .

How can researchers troubleshoot weak or absent signals when using ATL18 antibody?

When encountering weak or absent signals with ATL18 antibody, systematic troubleshooting should include: 1) Verifying protein extraction efficiency through total protein staining (Ponceau S or Coomassie); 2) Increasing protein concentration by loading more sample or concentrating extracts; 3) Optimizing antibody concentration by testing lower dilutions (1:500 or 1:250); 4) Extending primary antibody incubation time (overnight at 4°C); 5) Enhancing detection sensitivity by using amplification systems such as biotin-streptavidin; 6) Checking expression levels of ATL18 under different conditions, as it may be developmentally regulated or stress-induced; and 7) Testing different blocking agents (BSA vs. non-fat milk) to reduce background interference. Documentation of systematically varied parameters is essential for method optimization .

How can ATL18 antibody be utilized in studies examining plant stress responses and protein degradation pathways?

ATL18 antibody serves as a valuable tool for investigating stress-mediated protein degradation mechanisms in plants. For stress response studies, researchers can design time-course experiments exposing plants to different stressors (drought, salinity, pathogen elicitors, etc.) and use the antibody to monitor ATL18 protein accumulation, post-translational modifications, or subcellular localization changes. To study protein degradation pathways, the antibody can be employed in co-immunoprecipitation assays to identify ATL18 interaction partners or substrates that may be targeted for ubiquitination. Additionally, researchers can combine ATL18 immunodetection with proteasome inhibitors (MG132) to assess the role of ATL18 in protein turnover during specific developmental stages or stress conditions. These approaches provide mechanistic insights into how E3 ligases like ATL18 regulate plant adaptation to environmental challenges .

What approaches can be used to study post-translational modifications of ATL18 using the available antibody?

Studying post-translational modifications (PTMs) of ATL18 requires sophisticated methodological approaches. Researchers can employ the following strategies: 1) Two-dimensional gel electrophoresis followed by Western blotting with ATL18 antibody to detect charge shifts indicative of phosphorylation, ubiquitination, or other modifications; 2) Immunoprecipitation with ATL18 antibody followed by mass spectrometry analysis to identify specific modification sites; 3) Combination of ATL18 antibody with modification-specific antibodies (anti-phospho, anti-ubiquitin) in sequential immunoprecipitation to confirm modification types; and 4) Pharmacological treatments with PTM inhibitors or enhancers followed by Western blot analysis to assess changes in ATL18 migration patterns. These approaches help elucidate the regulatory mechanisms controlling ATL18 activity and stability in different physiological contexts .

How can ATL18 antibody be adapted for immunolocalization studies in plant tissues?

While the ATL18 antibody is primarily validated for Western blot and ELISA applications, researchers can optimize protocols for immunolocalization studies with careful method development. For immunohistochemistry or immunofluorescence in plant tissues, consider: 1) Tissue fixation with 4% paraformaldehyde or ethanol-acetic acid fixatives optimized for plant cell wall penetration; 2) Antigen retrieval steps such as enzymatic digestion with cellulase and pectinase to improve antibody accessibility; 3) Extended blocking (2-3 hours) with 5% BSA and 0.3% Triton X-100 to reduce non-specific binding; 4) Higher antibody concentrations (1:50 to 1:200) and longer incubation times (overnight at 4°C); 5) Amplification systems like tyramide signal amplification for low-abundance proteins; and 6) Rigorous controls including pre-immune serum and peptide competition controls. Preliminary testing on different tissue types and fixation methods will be necessary to establish reliable immunolocalization protocols .

How does ATL18 antibody performance compare with antibodies against other ATL family proteins?

When comparing the performance of ATL18 antibody with antibodies against other ATL family proteins, researchers should consider several factors: 1) Epitope specificity – ATL18 antibody targets unique regions of the protein, minimizing cross-reactivity with other ATL family members that share conserved domains; 2) Validation breadth – while some commercial ATL family antibodies have been validated across multiple applications, the current ATL18 antibody has confirmed specificity for Western blot and ELISA applications; 3) Signal-to-noise ratio – polyclonal antibodies like the ATL18 antibody typically offer robust signal detection but may have higher background compared to monoclonal alternatives for other ATL proteins. Researchers investigating multiple ATL family members should perform side-by-side comparisons, including epitope mapping and cross-reactivity tests, to accurately interpret comparative expression data .

What statistical approaches are recommended for quantifying ATL18 protein levels in comparative studies?

For rigorous quantitative analysis of ATL18 protein levels in comparative studies, researchers should implement the following statistical approaches: 1) Normalization to multiple reference proteins (not just one housekeeping gene) to account for potential regulatory changes under experimental conditions; 2) Technical replicates (minimum of three) and biological replicates (minimum of three independent experiments) to assess reproducibility; 3) Appropriate statistical tests based on data distribution – parametric tests (t-test, ANOVA) for normally distributed data or non-parametric alternatives (Mann-Whitney, Kruskal-Wallis) if normality cannot be assumed; 4) Multiple testing correction (e.g., Bonferroni or Benjamini-Hochberg) when performing numerous comparisons; and 5) Effect size calculations to determine biological significance beyond statistical significance. Quantification software should use background subtraction methods appropriate for plant tissue samples, which often have higher autofluorescence and non-specific binding .

How can researchers integrate ATL18 protein data with transcriptomic datasets to gain comprehensive insights?

Integrating ATL18 protein data with transcriptomic analyses requires methodological approaches that account for the different regulatory layers: 1) Time-course design capturing both transcript and protein measurements from the same samples, acknowledging potential temporal offsets between mRNA and protein changes; 2) Correlation analysis using Spearman or Pearson coefficients to identify relationships between ATL18 transcript abundance and protein levels across conditions; 3) Integration with public Arabidopsis expression databases (e.g., Genevestigator, BAR) to contextualize findings within broader expression patterns; 4) Network analysis incorporating known interaction partners and co-expressed genes to identify potential regulatory modules; and 5) Visualization approaches such as heat maps or principal component analysis to identify patterns across multiple datasets. This multi-omics approach helps distinguish between transcriptional and post-transcriptional regulatory mechanisms affecting ATL18 function under different experimental conditions .

How can ATL18 antibody be utilized in studies investigating plant immune responses?

The ATL18 antibody offers valuable opportunities for investigating plant immune signaling pathways. Researchers can design experiments to: 1) Monitor ATL18 protein accumulation following PAMP (Pathogen-Associated Molecular Pattern) treatment or pathogen infection using time-course Western blot analysis; 2) Compare ATL18 protein levels between wild-type plants and immune signaling mutants to position ATL18 within defense response pathways; 3) Perform co-immunoprecipitation with ATL18 antibody followed by mass spectrometry to identify potential immune-related substrates targeted for ubiquitination; 4) Use the antibody in conjunction with cell fractionation to track ATL18 subcellular relocalization during immune responses; and 5) Combine ATL18 protein analysis with ubiquitinome profiling to identify global changes in protein degradation patterns during plant defense responses. These approaches help elucidate the regulatory role of E3 ligases like ATL18 in controlling immune protein homeostasis .

What considerations should researchers take when adapting ATL18 antibody for chromatin immunoprecipitation (ChIP) experiments?

While the ATL18 antibody is not specifically validated for ChIP applications, researchers interested in examining potential chromatin associations would need to consider: 1) Thorough validation using tagged ATL18 constructs and ChIP-grade antibodies against the tag as positive controls; 2) Extensive optimization of crosslinking conditions, as plant tissues require modified fixation protocols compared to animal cells; 3) Sonication parameter adjustment to account for plant cell wall components that may interfere with chromatin shearing; 4) Implementation of stringent washing steps to reduce plant-specific background; 5) Quantification by qPCR targeting promoter regions of known or predicted ATL18-regulated genes; and 6) Rigorous negative controls including non-specific IgG and chromatin from atl18 knockout plants. Given that E3 ligases like ATL18 may have indirect chromatin associations through protein-protein interactions rather than direct DNA binding, researchers should consider alternative approaches such as proximity ligation assays to confirm spatial associations with chromatin .

How can proteomics approaches be combined with ATL18 antibody to identify potential ubiquitination targets?

To identify potential ubiquitination targets of ATL18, researchers can implement integrated proteomics strategies: 1) Immunoprecipitation with ATL18 antibody followed by mass spectrometry analysis to identify interacting proteins; 2) Comparative ubiquitinome analysis between wild-type and atl18 mutant plants to identify differentially ubiquitinated proteins; 3) Pulse-chase experiments with proteasome inhibitors to capture short-lived proteins stabilized in atl18 mutants; 4) Validation of candidate targets using in vitro ubiquitination assays with recombinant ATL18 protein; and 5) In vivo confirmation through co-immunoprecipitation and bimolecular fluorescence complementation. The experimental design should include appropriate controls to distinguish direct targets from indirect effects and account for the dynamic nature of ubiquitination processes. This multi-layered approach helps construct a comprehensive picture of ATL18's role in protein homeostasis networks regulating plant development and stress responses .

What emerging technologies might enhance the utility of ATL18 antibody in plant research?

Emerging technologies that could significantly enhance ATL18 antibody applications include: 1) Single-cell proteomics methods adapted for plant tissues, allowing analysis of cell-specific ATL18 expression patterns; 2) Microfluidic antibody-based protein detection systems for high-throughput, low-volume sample analysis; 3) Organelle-specific proximity labeling techniques (BioID, APEX) combined with ATL18 antibody validation to map spatiotemporal protein interactions; 4) CRISPR-engineered epitope-tagged ATL18 lines for enhanced detection specificity; 5) Antibody-based biosensors for real-time monitoring of ATL18 conformational changes or activity in living plant cells; and 6) Advanced super-resolution microscopy techniques optimized for plant tissues to visualize subcellular localization at nanometer resolution. These technologies promise to overcome current limitations in studying dynamic protein functions in complex plant systems, providing unprecedented insights into E3 ligase biology .

How might ATL18 antibody research inform agricultural applications and crop improvement strategies?

Research utilizing ATL18 antibody has potential agricultural implications as it helps elucidate fundamental mechanisms of plant stress response and development regulation. Findings may inform crop improvement strategies through: 1) Identification of orthologous E3 ligases in crop species that could be targets for breeding or biotechnological approaches; 2) Development of diagnostic tools using antibodies against ATL family proteins to monitor stress response activation in field conditions; 3) Understanding of protein degradation networks that regulate agronomically important traits such as flowering time, senescence, or disease resistance; 4) Characterization of ubiquitination pathways that influence yield stability under adverse environmental conditions; and 5) Identification of potential targets for gene editing to enhance stress resilience. By connecting basic research on protein quality control mechanisms to applied agricultural challenges, ATL18 research contributes to the development of next-generation crop improvement strategies .