AOS1 Antibody

What is AOS1 and what cellular functions does it perform?

AOS1 (also known as SAE1, SUA1, UBLE1A) is a 38kDa protein that functions as the SUMO-activating enzyme subunit 1 in the SUMOylation pathway . It forms a heterodimer with UBA2 to create the E1 SUMO-activating enzyme complex that initiates the SUMO conjugation cascade. This post-translational modification system regulates protein function, localization, and stability across numerous cellular processes including transcriptional regulation, DNA repair, and cell cycle progression. AOS1 is ubiquitously expressed in human, mouse, and rat tissues, making it an important target for comparative studies across model organisms . The protein contains several functional domains that facilitate ATP binding and SUMO activation, which are essential for its enzymatic function in the first step of the SUMOylation pathway.

What applications are available for AOS1 antibodies in research settings?

AOS1 antibodies have been validated for multiple research applications with varying sensitivities and specificities. Based on current research protocols, the primary applications include:

These applications allow researchers to detect endogenous levels of total AOS1 protein in various experimental contexts . When selecting an application, consider the specific sensitivity requirements and sample type to achieve optimal results.

How should I optimize Western blot protocols for AOS1 detection?

For optimal AOS1 detection via Western blot, follow these methodological considerations:

• Sample preparation: Use RIPA or NP-40 lysis buffers supplemented with protease inhibitors. For SUMOylation studies, include 20mM N-ethylmaleimide to prevent SUMO deconjugation during lysis.

• Protein loading: Load 20-40μg of total protein per lane for cell lysates. For tissue samples, optimization may be required depending on AOS1 expression levels.

• Antibody dilutions: Use Anti-AOS1 antibodies at 1:500-1:1000 dilution in 5% BSA or non-fat milk in TBST . This range has been validated in multiple cell lines including 293, HepG2, and Colo cells .

• Detection controls: Include positive controls such as 293 cell lysates, which show consistent AOS1 expression. For specificity validation, perform peptide competition assays with the immunizing peptide, which should block the 38kDa band corresponding to AOS1 .

• Troubleshooting: If experiencing high background, increase blocking time (1-2 hours) and washing steps. For weak signals, extend exposure time or increase antibody concentration, ensuring you stay within the validated range.

Western blot validation data from multiple studies confirms that the appropriate antibody will detect a single 38kDa band in human, mouse, and rat samples, with this band being eliminated in peptide competition assays, confirming specificity .

What are the recommended storage conditions for maintaining AOS1 antibody activity?

Proper storage is critical for maintaining antibody functionality and preventing degradation. For AOS1 antibodies:

• Short-term storage (up to 1 week): Store at 4°C in the original buffer conditions. Most commercial AOS1 antibodies are shipped at 4°C for short-term stability .

• Long-term storage: Store at -20°C in small aliquots to prevent repeated freeze-thaw cycles. The antibodies are typically supplied in Phosphate Buffered Saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% Sodium Azide, and 50% Glycerol, which helps maintain stability during freeze-thaw .

• Freeze-thaw cycles: Minimize freeze-thaw cycles as each cycle can reduce antibody activity by approximately 10-15%. Make multiple small aliquots upon first thawing to prevent repeated freezing of the entire stock .

• Working solution: Diluted antibody solutions should be prepared fresh and used within 24 hours for optimal performance. If storage is necessary, keep at 4°C for no more than 5 days.

• Stability testing: For critical experiments, periodically test antibody activity on positive control samples to ensure continued functionality, especially after extended storage periods.

Following these storage recommendations will maintain antibody specificity and sensitivity for your experimental applications.

How can I validate the specificity of AOS1 antibodies in my experimental model?

Thorough validation of AOS1 antibody specificity is essential for reliable research outcomes. Implement these comprehensive validation strategies:

• Peptide competition assay: Pre-incubate the AOS1 antibody with the immunizing peptide (synthetic peptide derived from human AOS1, typically amino acids 221-270), then perform Western blot analysis. The specific 38kDa band should be significantly reduced or eliminated . This has been demonstrated in validation studies with 293 cell lysates where the AOS1 signal was blocked by the synthesized immunogen peptide .

• Genetic validation: Use CRISPR/Cas9 or siRNA knockdown of AOS1 expression. Compare antibody staining in wildtype versus knockdown samples across all intended applications. A specific antibody will show reduced signal proportional to the knockdown efficiency.

• Multiple antibody comparison: Test antibodies raised against different epitopes of AOS1 (such as those targeting internal regions versus N or C-terminal domains). Consistent detection patterns across different antibodies increase confidence in specificity.

• Cross-species reactivity assessment: If working with non-human models, validate the antibody in your specific species. While most commercial AOS1 antibodies react with human, mouse, and rat samples , cross-reactivity with other species requires experimental confirmation.

• Mass spectrometry validation: For the most rigorous validation, perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody.

• Application-specific controls: For each application (WB, IF, ELISA), include appropriate negative controls (secondary antibody only, isotype control) and positive controls (cell lines with known AOS1 expression).

What are the considerations for using AOS1 antibodies in quantitative analysis of SUMOylation pathways?

Quantitative analysis of AOS1 in SUMOylation pathways requires careful experimental design:

• Normalization strategy: When quantifying AOS1 levels, normalize to appropriate housekeeping proteins (GAPDH, β-actin) that remain stable under your experimental conditions. For Cell-ELISA approaches, parallel GAPDH detection can serve as an internal control .

• Dynamic range determination: Establish the linear dynamic range of AOS1 detection for your specific antibody and application. This typically requires a standard curve using recombinant AOS1 protein or cell lysates with known AOS1 expression levels.

• SUMOylation dynamics: When studying AOS1 in the context of SUMOylation, consider that while AOS1 protein levels may remain relatively stable, its activity can be modulated by post-translational modifications or protein-protein interactions. Therefore, complement antibody-based detection of AOS1 with functional assays of SUMO E1 activity.

• Co-immunoprecipitation approaches: For studying AOS1-UBA2 heterodimer formation or interaction with SUMO proteins, use co-immunoprecipitation protocols optimized to preserve native protein interactions. This typically involves milder lysis conditions (NP-40 based buffers) compared to standard Western blot sample preparation.

• Stimulation experiments: When analyzing AOS1 involvement in response to cellular stress (heat shock, osmotic stress, etc.), carefully time-match samples and controls to account for potential temporal dynamics in the SUMOylation pathway.

• Multiplexed detection: When possible, use multiplexed detection methods to simultaneously analyze multiple components of the SUMOylation pathway (AOS1, UBA2, SUMO1/2/3) to gain insights into pathway coordination.

These methodological considerations ensure robust quantitative analysis of AOS1 in the broader context of SUMOylation pathways.

How do different epitopes of AOS1 affect antibody binding and experimental outcomes?

The epitope recognized by an AOS1 antibody significantly impacts experimental results through several mechanisms:

When selecting an AOS1 antibody, carefully review the immunogen information and validation data to ensure the epitope recognition profile matches your experimental requirements.

What are the technical considerations for using AOS1 antibodies in immunofluorescence studies?

For successful immunofluorescence detection of AOS1, consider these technical parameters:

• Fixation method: For AOS1 detection, paraformaldehyde fixation (4%, 15-20 minutes at room temperature) preserves protein antigenicity while maintaining cellular architecture. Methanol fixation may be used for certain epitopes but can affect some conformational epitopes.

• Permeabilization: Use 0.1-0.2% Triton X-100 in PBS for 5-10 minutes to allow antibody access to intracellular AOS1. Overly harsh permeabilization can disrupt nuclear architecture where AOS1 is often concentrated.

• Optimal antibody dilution: For immunofluorescence, use AOS1 antibodies at 1:100-1:500 dilution, which is typically more concentrated than for Western blot applications . Always include a titration series in preliminary experiments to determine optimal signal-to-noise ratio.

• Blocking: Use 5-10% normal serum (from the species of the secondary antibody) with 1% BSA to minimize background. Extended blocking (1-2 hours at room temperature) improves signal specificity.

• Nuclear counterstaining: Since AOS1 can have nuclear localization, select nuclear counterstains (DAPI, Hoechst) that don't overlap with your fluorophore-conjugated secondary antibody.

• Co-localization studies: For studying AOS1 interaction with SUMOylation machinery, consider dual immunofluorescence with antibodies against UBA2 or SUMO proteins. Ensure primary antibodies are from different host species to avoid cross-reactivity.

• Controls: Include both positive controls (cell lines with known AOS1 expression patterns) and negative controls (secondary antibody only, peptide competition) . For rigorous validation, include AOS1 knockdown cells as biological negative controls.

These technical considerations ensure specific and reproducible detection of AOS1 in immunofluorescence applications.

How can I troubleshoot inconsistent results when using AOS1 antibodies?

When troubleshooting inconsistent AOS1 antibody results, systematically evaluate these common sources of variability:

• Antibody quality: Antibody degradation can occur with improper storage or excessive freeze-thaw cycles. Aliquot antibodies upon receipt and store at -20°C. Test antibody performance on positive control samples (e.g., 293 cells) that consistently express AOS1 .

• Sample preparation: Inconsistent lysis conditions can affect AOS1 detection. Standardize lysis buffers, protein extraction methods, and protein quantification techniques. For Western blot, ensure complete protein denaturation by adequate heating in sample buffer.

• Protocol variations: Minor protocol modifications can significantly impact results. Document and standardize all parameters, including blocking conditions, antibody incubation times/temperatures, and washing stringency.

• Biological variability: AOS1 expression or localization may vary with cell cycle stage, cell confluence, or stress conditions. Standardize culture conditions and cell harvesting protocols to minimize these variables.

• Post-translational modifications: AOS1 function in the SUMOylation pathway may be regulated by post-translational modifications that affect antibody recognition. Consider using multiple antibodies targeting different epitopes to provide complementary information.

• Cross-reactivity: Despite high specificity, some AOS1 antibodies may exhibit cross-reactivity with similar proteins. Validate specificity through peptide competition assays or genetic approaches (siRNA knockdown) .

• Detection system sensitivity: Different secondary antibodies and detection systems have varying sensitivities. Standardize these components across experiments and consider signal amplification methods for low-abundance samples.

Systematic evaluation of these factors will help identify the source of inconsistency and establish reliable AOS1 detection protocols.

What are the latest approaches for studying AOS1-dependent SUMOylation using antibody-based techniques?

Advanced antibody-based techniques for studying AOS1-dependent SUMOylation include:

• Proximity Ligation Assay (PLA): This technique allows visualization of AOS1 interactions with UBA2 or SUMO substrates at the single-molecule level in fixed cells. PLA provides spatial resolution of interactions and can detect transient associations that may be missed by co-immunoprecipitation.

• FRET-based immunoassays: By labeling anti-AOS1 and anti-UBA2 antibodies with appropriate FRET pairs, researchers can monitor the formation and disassembly of the SUMO E1 complex in real-time in response to various stimuli.

• Chromatin Immunoprecipitation (ChIP): Modified ChIP protocols using AOS1 antibodies can identify genomic regions where AOS1-dependent SUMOylation machinery is recruited, particularly relevant for understanding SUMOylation in transcriptional regulation.

• Single-cell antibody-based analysis: Advanced imaging and flow cytometry techniques using AOS1 antibodies can reveal cell-to-cell variability in AOS1 expression and localization, providing insights into heterogeneous responses to SUMOylation-inducing stimuli.

• Sequential immunoprecipitation: This approach involves first immunoprecipitating with anti-SUMO antibodies followed by anti-AOS1 antibodies (or vice versa) to identify complexes containing both proteins, helping to distinguish AOS1's various functional pools.

• Multiplexed imaging: Combining AOS1 antibodies with antibodies against multiple SUMOylation components in highly multiplexed imaging platforms enables comprehensive pathway analysis in intact cells or tissues.

• Antibody-based biosensors: Engineering antibody fragments derived from AOS1 antibodies into fluorescent biosensors allows monitoring of AOS1 conformational changes or interactions in living cells.

These advanced approaches leverage the specificity of AOS1 antibodies to provide deeper insights into SUMOylation dynamics in various cellular contexts.

How can I use AOS1 antibodies in combination with other tools to study global SUMOylation patterns?

Integrating AOS1 antibodies with complementary techniques provides comprehensive insights into SUMOylation:

• Antibody-substrate linkage analysis: Combine AOS1 antibodies with antibodies against specific SUMO isoforms (SUMO1, SUMO2/3) in sequential immunoprecipitation experiments to identify which SUMO pathways are most dependent on AOS1 activity.

• Pharmacological modulation: Use AOS1 antibodies to monitor pathway responses to SUMO E1 inhibitors (such as ML-792), helping differentiate between direct effects on AOS1-dependent processes versus compensatory mechanisms.

• Mass spectrometry integration: Immunoprecipitate with AOS1 antibodies followed by mass spectrometry to identify the complete interactome of AOS1 under various cellular conditions, revealing potential regulatory proteins beyond the core SUMOylation machinery.

• Cellular stress response profiling: Apply AOS1 antibodies in cellular assays following various stressors (heat shock, oxidative stress, hypoxia) to quantify stress-specific changes in AOS1 localization, complex formation, or post-translational modifications.

• CRISPR screening follow-up: After genome-wide CRISPR screens for SUMOylation-dependent phenotypes, use AOS1 antibodies to validate hits by assessing how genetic perturbations affect AOS1 expression, localization, or activity.

• Computational modeling: Incorporate quantitative AOS1 antibody data from multiple experimental conditions into computational models of the SUMOylation pathway to predict system-level responses to perturbations.

• Single-cell correlation analysis: Combine single-cell immunofluorescence data from AOS1 antibodies with data from other SUMOylation components to identify cell-to-cell variations and potential regulatory relationships within the pathway.

This integrative approach leverages AOS1 antibodies as part of a broader experimental strategy to understand the complex regulation and diverse functions of the SUMOylation pathway.

What considerations should be made when developing customized AOS1 antibodies for specific research applications?

Researchers developing customized AOS1 antibodies should consider:

• Epitope selection: Target unique regions of AOS1 that are not conserved in related proteins to minimize cross-reactivity. Careful bioinformatic analysis of AOS1 sequence (including post-translational modification sites and protein interaction domains) is essential for optimal epitope selection .

• Application-specific design: Different applications require antibodies with different properties. For Western blot, linear epitopes are suitable; for immunoprecipitation or ChIP, surface-exposed epitopes are preferable; for conformational studies, discontinuous epitopes may be required .

• Cross-species requirements: If the antibody needs to recognize AOS1 across multiple species, target epitopes with high sequence conservation. Alternatively, if species-specific detection is required, select divergent regions .

• Validation strategy: Plan comprehensive validation experiments including peptide competition, knockdown/knockout controls, and comparison with existing commercial antibodies. For rigorous validation, consider mass spectrometry to confirm antibody specificity .

• Affinity requirements: Higher affinity is generally beneficial, but extremely high affinity can sometimes increase non-specific binding. Balance affinity optimization with specificity requirements based on your application's sensitivity needs .

• Signal-to-noise optimization: For challenging applications like immunohistochemistry or live-cell imaging, prioritize antibodies with exceptional signal-to-noise properties through careful screening and selection processes .

• Production scale: Consider whether the antibody will be needed for long-term projects requiring consistent lots over time, which might favor monoclonal development despite higher initial costs.

Recent advances in computational antibody design can significantly enhance these traditional approaches by predicting optimal epitopes and binding characteristics before experimental production .