ATG10 Antibody

What is ATG10 and why is it significant in autophagy research?

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.

What types of ATG10 antibodies are available for research applications?

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

What are the validated applications for ATG10 antibodies?

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

• Immunocytochemistry (ICC): For cultured cells

• ELISA: For quantitative measurement

• Flow cytometry (FACS): For cell population analysis

Application-specific dilutions vary by antibody; for example, IHC-P typically requires 1:50-1:100 dilution for optimal results .

How should I validate ATG10 antibody specificity?

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.

What are the optimal sample preparation methods for Western blotting with ATG10 antibodies?

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.

How do I select the appropriate ATG10 antibody for my specific research application?

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.

How can ATG10 antibodies be used to study the dynamics of autophagosome formation?

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.

What are the best practices for analyzing ATG10 isoform expression with antibodies?

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.

How can I develop quantitative assays for measuring ATG10 activity using antibodies?

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.

How do I address non-specific binding and background issues with ATG10 antibodies?

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:

  • Include ATG10 knockout/knockdown samples to identify non-specific bands

  • Use competing peptides that block antibody binding to confirm specificity

  • Perform secondary-only controls to identify background from secondary antibodies

• 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.

Why might Western blot results with ATG10 antibodies differ from immunofluorescence/IHC data?

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:

  • Verify antibody has been validated for each specific application

  • Use appropriate positive controls for each method

When encountering discrepancies, parallel validation using genetic approaches (siRNA, CRISPR) can help resolve which method accurately reflects ATG10 biology.

How can I optimize antibody detection of ATG10 in tissues with low expression levels?

For tissues with low ATG10 expression:

• Signal amplification strategies:

  • Use tyramide signal amplification (TSA) for IHC/IF

  • Employ highly sensitive chemiluminescent substrates for WB

  • Consider biotin-streptavidin amplification systems

• 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.

How can ATG10 antibodies be applied in studying disease mechanisms?

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:

  • Analysis of ATG10 modulation during pathogen infection

  • Investigation of pathogen proteins targeting the ATG10-mediated conjugation system

  • Study of adenovirus-mediated cell lysis mechanisms involving ATG10

• 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.

What novel techniques are emerging for studying ATG10 using antibody-based approaches?

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.

How can I determine the functional consequences of ATG10 post-translational modifications using antibodies?

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.

What are the optimal storage conditions for maintaining ATG10 antibody performance?

To maintain optimal antibody performance:

• Storage temperature recommendations:

  • Store lyophilized antibodies at -20°C to -70°C before reconstitution

  • Store reconstituted antibodies at -20°C to -70°C for long-term storage (up to 6 months)

  • Short-term storage (up to 1 month) at 2-8°C is acceptable for reconstituted antibodies under sterile conditions

• 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:

  • Minimize freeze-thaw cycles as they can degrade antibody quality

  • Allow antibodies to equilibrate to room temperature before opening

  • Use a manual defrost freezer for storage

• 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.

What quality control measures should I implement for long-term ATG10 antibody studies?

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.