ATG10 (Autophagy Related 10) functions as an E2-like enzyme that catalyzes the conjugation of ATG12 to ATG5, a critical step in autophagosome formation. This ATG5:ATG12 heterodimer subsequently associates non-covalently with ATG16 multimers to generate autophagosomes . As a key component of the core autophagy machinery, ATG10 serves as an ATG5-recognition molecule and plays a role in adenovirus-mediated cell lysis . Human ATG10 is 220 amino acids in length with an active site at Cys166 that forms a thiol ester bond with the C-terminal Gly of ATG12 . Due to its central role in autophagy regulation, studying ATG10 provides valuable insights into fundamental cellular degradation and recycling mechanisms.
Multiple types of ATG10 antibodies are available for research applications:
Host species: Predominantly rabbit-derived, but also available from sheep and mouse hosts
Clonality: Both polyclonal (e.g., ABIN6244154) and monoclonal (e.g., EPR4804) options exist
Target regions: Antibodies targeting different epitopes including N-terminal regions (AA 15-45), C-terminal regions, and full-length protein
Format variations: Available as unconjugated antibodies or conjugated with various labels including biotin, PE, PerCP, Atto 488, and Atto 390 for specialized applications
ATG10 antibodies have been validated for multiple experimental applications:
Western blotting (WB): The predominant application, detecting ATG10 at approximately 28 kDa
Immunohistochemistry (IHC): Both paraffin-embedded (IHC-P) and frozen sections (IHC-F)
Immunofluorescence (IF): For subcellular localization studies
Application-specific dilutions vary by antibody; for example, IHC-P typically requires 1:50-1:100 dilution for optimal results .
Rigorous validation of ATG10 antibody specificity should involve multiple complementary approaches:
Knockout validation: Use ATG10 knockout cell lines (such as the Human ATG10 knockout HeLa cell line) as a negative control to confirm signal disappearance
Knockdown validation: Compare signal between wild-type and siRNA/shRNA-mediated knockdown samples
Multiple antibody approach: Verify signals using different antibodies targeting distinct epitopes of ATG10
Molecular weight confirmation: Ensure the detected band corresponds to the expected molecular weight (~28 kDa for human ATG10)
Positive controls: Include cell lines with established ATG10 expression (e.g., A431, HeLa, HEK293)
Documentation of antibody validation using these approaches significantly strengthens research reliability and reproducibility.
For optimal Western blot results with ATG10 antibodies:
Cell lysis: Use specialized buffers containing phosphatase and protease inhibitors to prevent post-lysis degradation
Protein quantification: Normalize samples to ensure equal loading
Separation conditions: Use 10-12% SDS-PAGE gels for optimal resolution around the 28 kDa region
Transfer parameters: PVDF membranes have been validated for successful ATG10 detection
Blocking: Use 5% non-fat milk or BSA in TBST
Antibody dilution: For example, 1 μg/mL of antibody has been successful in detecting ATG10 in carcinoma and embryonic kidney cell lines
Buffer systems: Immunoblot Buffer Group 2 has been validated for some ATG10 antibodies
Attention to these methodological details significantly enhances detection sensitivity and specificity.
Selection criteria should include:
Target species concordance: Ensure the antibody has been validated for your experimental species (human, mouse, rat)
Application validation: Confirm the antibody has been tested for your specific application (WB, IHC, IF, etc.)
Epitope consideration: For domain-specific studies, select antibodies targeting specific regions (N-terminal vs. C-terminal)
Clonality requirements: Choose monoclonal antibodies for higher specificity or polyclonal antibodies for broader epitope recognition
Conjugation needs: Select appropriately labeled antibodies for applications requiring fluorescence or enzymatic detection
Validation robustness: Prioritize antibodies validated with knockout controls and multiple cell lines
Matching these criteria to your experimental requirements is essential for generating reliable data.
Advanced approaches to study autophagosome formation dynamics include:
Temporal immunofluorescence: Use ATG10 antibodies in conjunction with other autophagy markers (LC3, p62) at defined time points after autophagy induction
Co-immunoprecipitation (Co-IP): Use ATG10 antibodies to pull down protein complexes and identify interaction partners during different stages of autophagosome formation
Proximity ligation assay (PLA): Combine ATG10 antibodies with antibodies against potential interacting proteins to visualize protein complexes in situ
FRET/FLIM analysis: Use fluorescently conjugated ATG10 antibodies in combination with labeled ATG5/ATG12 antibodies to analyze protein proximity
Super-resolution microscopy: Apply ATG10 antibodies in techniques like STORM or PALM for nanoscale localization during autophagosome biogenesis
These approaches provide mechanistic insights into the spatiotemporal dynamics of ATG10's role in autophagy.
Multiple ATG10 isoform variants exist, including:
A variant with deletion of amino acids 37-72
A variant with an alternate start site at Met43 and a 39 amino acid substitution for aa 152-220
A variant showing a 53 amino acid substitution for aa 73-220
To effectively analyze these isoforms:
Epitope mapping: Select antibodies whose epitopes can distinguish between isoforms
Isoform-specific controls: Generate positive controls for each isoform using overexpression systems
High-resolution gel systems: Use gradient gels or Phos-tag™ gels for better separation of similar-sized isoforms
Mass spectrometry validation: Confirm antibody-detected isoforms using proteomic approaches
RT-PCR correlation: Correlate protein detection with transcript levels of specific isoforms
This multi-faceted approach enables accurate characterization of ATG10 isoform expression patterns in different cellular contexts.
Quantitative assays for ATG10 E2-like enzyme activity include:
ATG5-ATG12 conjugation assay: Measure the formation of ATG5-ATG12 conjugates using specific antibodies against each protein
In vitro conjugation system: Develop a purified protein system with recombinant ATG10, ATG5, and ATG12, using antibodies to detect conjugation products
FRET-based activity reporters: Design systems where ATG10-mediated conjugation alters FRET efficiency
Split-luciferase complementation: Engineer ATG5 and ATG12 constructs that generate luciferase activity upon ATG10-mediated conjugation
Phosphorylation-specific antibodies: Develop antibodies that recognize post-translational modifications of ATG10 that correlate with its activity state
These assays provide functional readouts beyond simple expression analysis, offering insights into the enzymatic activity of ATG10 in various experimental conditions.
When encountering high background or non-specific binding:
Optimization strategies:
Titrate antibody dilutions (e.g., test ranges from 1:500 to 1:2000 for WB)
Evaluate different blocking agents (BSA vs. milk vs. commercial blockers)
Test longer washing times and increased detergent concentrations in wash buffers
Consider alternative secondary antibodies
Validation controls:
Signal enhancement approaches:
Use signal amplification systems selectively for true signal enhancement
Consider antigen retrieval optimization for IHC applications
Evaluate different detection systems (chemiluminescence vs. fluorescence)
These systematic approaches help distinguish true ATG10 signal from artifacts.
Discrepancies between detection methods often stem from:
Epitope accessibility differences:
Protein denaturation in WB may expose epitopes hidden in native conformation
Fixation methods in IF/IHC may mask certain epitopes
Cross-reactivity considerations:
Different techniques may reveal cross-reactivity with structurally similar proteins
Confirm findings using multiple antibodies targeting different epitopes
Sensitivity thresholds:
Western blotting may detect low abundance proteins missed by IF/IHC
Signal amplification differences between methods affect detection limits
Methodological validation:
When encountering discrepancies, parallel validation using genetic approaches (siRNA, CRISPR) can help resolve which method accurately reflects ATG10 biology.
For tissues with low ATG10 expression:
Signal amplification strategies:
Sample enrichment approaches:
Immunoprecipitate ATG10 before Western blotting to concentrate the protein
Use tissue microdissection to isolate regions with higher expression
Consider cell sorting to enrich for cell populations with higher expression
Detection optimization:
Increase antibody concentration and incubation time
Reduce washing stringency while maintaining specificity
Use fluorescent secondary antibodies with minimal autofluorescence wavelengths
Alternative validation:
Correlate protein detection with mRNA expression data
Consider using genetic models with ATG10 overexpression as positive controls
These approaches can help detect physiologically relevant low-level expression of ATG10.
ATG10 antibodies enable investigation of autophagy dysregulation in various diseases:
Cancer research applications:
Tissue microarray analysis correlating ATG10 expression with clinical outcomes
Monitoring ATG10 changes during treatment response
Analysis of ATG10 in drug-resistant vs. sensitive cell populations
Neurodegenerative disease applications:
Co-localization studies with disease-specific protein aggregates
Temporal analysis of ATG10 expression during disease progression
Examination of ATG10 in animal models of neurodegeneration
Infectious disease applications:
Metabolic disease applications:
Examination of ATG10 expression in response to metabolic stressors
Analysis of tissue-specific ATG10 regulation in metabolic disorders
These applications provide insights into disease mechanisms and potential therapeutic targets.
Emerging techniques include:
Single-cell protein analysis:
Mass cytometry (CyTOF) with ATG10 antibodies for single-cell profiling
Microfluidic antibody capture for single-cell Western blotting
Advanced imaging approaches:
Lattice light-sheet microscopy for high-speed 3D imaging of ATG10 dynamics
Expansion microscopy to spatially resolve ATG10 in autophagosome formation
Correlative light and electron microscopy (CLEM) to connect ATG10 localization with ultrastructure
Spatial proteomics:
Multiplexed ion beam imaging (MIBI) for spatial analysis of multiple autophagy proteins
Digital spatial profiling for regional analysis of ATG10 in complex tissues
Proximity labeling techniques:
Antibody-guided APEX2 proximity labeling to identify ATG10 interaction networks
Split-TurboID systems for conditional proximity labeling of ATG10 complexes
These emerging approaches offer unprecedented insights into ATG10 biology in complex biological systems.
Strategies for studying ATG10 post-translational modifications include:
Modification-specific antibodies:
Phospho-specific antibodies targeting known or predicted ATG10 phosphorylation sites
Antibodies recognizing ubiquitinated, SUMOylated, or acetylated forms of ATG10
Analytical approaches:
2D gel electrophoresis followed by ATG10 immunoblotting to resolve modified forms
Immunoprecipitation with ATG10 antibodies followed by mass spectrometry analysis
Phos-tag™ gel electrophoresis to separate phosphorylated forms
Functional correlation studies:
Site-directed mutagenesis of modification sites combined with antibody detection
Correlation of modifications with ATG10 enzymatic activity in conjugation assays
Temporal analysis of modifications during autophagy induction and progression
Cell-based assays:
Treatment with modification-specific inhibitors followed by ATG10 immunoblotting
Co-localization of modified ATG10 with autophagosome formation markers
These approaches connect post-translational regulation of ATG10 with its functional roles in autophagy.
To maintain optimal antibody performance:
Storage temperature recommendations:
Aliquoting strategies:
Divide reconstituted antibodies into single-use aliquots
Use sterile techniques during aliquoting to prevent contamination
Use tubes with minimal adhesion to minimize antibody loss
Handling considerations:
Reconstitution practices:
Follow manufacturer-specific recommendations for diluents
Allow complete dissolution before aliquoting
Record reconstitution date on all aliquots
These practices help maintain antibody performance over time, ensuring consistent experimental results.
For long-term studies requiring consistent antibody performance:
Reference sample validation:
Create and store standard positive control lysates in aliquots
Run reference samples in each experimental batch
Document band intensity or signal-to-noise ratios for quantitative comparison
Lot-to-lot validation:
Test new antibody lots against old lots using identical samples
Document any sensitivity or specificity differences between lots
Maintain records of lot numbers used for each experiment
Periodic specificity testing:
Re-validate using knockout/knockdown controls periodically
Test for potential cross-reactivity with related proteins
Verify epitope integrity through peptide competition assays
Performance trending:
Monitor signal intensity and background levels over time
Track antibody dilution requirements for consistent results
Document any changes in antibody performance characteristics
Implementing these quality control measures ensures experimental consistency and facilitates troubleshooting when performance issues arise.