ATG7 Antibody

What is ATG7 and what cellular functions does it perform?

ATG7 (Autophagy-related gene 7) is an essential autophagy effector enzyme that plays critical roles in both autophagy-dependent and autophagy-independent cellular processes. In autophagy, ATG7 functions as an E1-like enzyme that activates ATG12 and LC3, facilitating autophagosome formation. ATG7 has been shown to induce basal autophagy by upregulating autophagic flux without increasing cytotoxicity . Beyond autophagy, ATG7 modulates p53 activity to regulate cell cycle progression and cellular survival during metabolic stress . It also interacts with and regulates proteins like PDCD4 through mechanisms independent of its autophagic functions .

In which experimental applications can ATG7 antibodies be utilized?

ATG7 antibodies have proven effective across multiple experimental applications:

• Western blotting: Detects ATG7 at approximately 75 kDa in human cell lines such as HeLa and HepG2

• Immunocytochemistry: Localizes ATG7 in cellular compartments, particularly autophagosomes

• Immunohistochemistry: Detects ATG7 in formalin-fixed paraffin-embedded tissue sections, showing localization in neuronal cell bodies and processes in human brain cortex samples

• Flow cytometry: Measures ATG7 expression levels in permeabilized cells

• Co-immunoprecipitation: Confirms protein-protein interactions involving ATG7

• Simple Western analysis: Automated capillary-based detection of ATG7

What are the known subcellular localizations of ATG7?

ATG7 demonstrates dynamic subcellular localization patterns depending on cellular context. Under basal conditions, ATG7 displays diffuse cytoplasmic distribution with stronger perinuclear staining when overexpressed . During autophagy induction, ATG7 can be visualized in proximity to developing autophagosomes. Immunofluorescence studies using specific antibodies have confirmed localization to autophagosomes in multiple cell types . Additionally, nuclear localization has been suggested in some contexts, particularly related to its non-autophagic functions, although cytoplasmic localization predominates in most cell types studied.

How are ATG7 expression and function altered in disease states?

ATG7 expression and function show significant alterations across various disease contexts:

What controls should be included when validating ATG7 antibody specificity?

When validating ATG7 antibody specificity, researchers should implement several control strategies:

• Positive controls: Include cell lines known to express ATG7, such as HeLa and HepG2 human cell lines

• Negative controls:

  • Use ATG7 knockout cell lines created via CRISPR-Cas9 as true negative controls

  • Include isotype control antibodies in flow cytometry and immunostaining experiments

• Peptide competition assays: Pre-incubate the antibody with recombinant ATG7 protein to verify signal suppression

• siRNA knockdown: Compare signal intensity between ATG7-depleted and control cells

• Multiple antibody validation: Use antibodies recognizing different epitopes of ATG7

• Cross-reactivity testing: Verify species specificity when working with human/mouse cross-reactive antibodies

Proper validation ensures experimental reliability and prevents misinterpretation of results, particularly in complex disease models where ATG7 expression may be altered.

What are optimal sample preparation methods for detecting ATG7 in different experimental contexts?

Sample preparation methods vary by experimental application:

For Western blotting:

• Use reducing conditions with standard RIPA or NP-40 lysis buffers containing protease inhibitors

• Sample preparation under Immunoblot Buffer Group 1 conditions has been validated

• Recommended loading: 20-30 μg total protein per lane

• Optimal antibody dilution: 2 μg/mL for primary antibody incubation

For immunofluorescence:

• Fixation: 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for adherent cells

• For non-adherent cells, follow specialized protocols for fluorescent ICC staining

• Primary antibody concentration: 25 μg/mL with overnight incubation at 4°C

For immunohistochemistry:

• Formalin-fixed paraffin-embedded sections require heat-induced epitope retrieval using basic antigen retrieval reagents

• Optimal antibody concentration: 15 μg/mL with overnight incubation at 4°C

For flow cytometry:

• Cell fixation with flow cytometry fixation buffer

• Permeabilization using specialized permeabilization/wash buffer

• Antibody concentration optimization is critical for reducing background signal

How can ATG7 antibodies be used to monitor autophagy flux?

ATG7 antibodies can be combined with other autophagy markers to effectively monitor autophagy flux:

• Dual marker approach: Combine ATG7 antibody detection with LC3-II and p62 antibodies. ATG7 overexpression increases LC3-II levels and decreases p62 accumulation, indicating enhanced autophagic flux .

• Autophagy inhibitor studies: Compare ATG7, LC3-II, and p62 levels before and after treatment with autophagy inhibitors:

  • Bafilomycin A1 or Chloroquine (lysosomal inhibitors) to assess flux completion

  • Monitor changes in the ATG5-ATG12 conjugate formation, which requires ATG7 activity

• Live-cell imaging: Combine ATG7 antibody staining with fluorescently-tagged LC3 to visualize autophagosome formation and clearance.

• Quantitative analysis:

  • Measure LC3 puncta formation in ATG7-overexpressing versus control cells

  • Perform densitometric analysis of Western blots for LC3-II/LC3-I ratio and p62 levels

  • Track autophagosome-lysosome fusion events using appropriate markers

This methodological approach has been validated in multiple studies, demonstrating that ATG7 overexpression induces autophagy in a dose-dependent manner, as measured by increased LC3-II conversion and autophagic flux .

How can ATG7 antibodies be used to investigate autophagy-independent functions of ATG7?

Investigating autophagy-independent functions of ATG7 requires specialized experimental designs:

• Differential inhibition strategy:

  • Use autophagy inhibitors (Bafilomycin A1, CQ) to block canonical autophagy

  • Observe persistent ATG7-dependent effects that occur despite autophagy inhibition

  • Monitor protein-protein interactions unique to ATG7's non-autophagic roles

• Protein interaction studies:

  • Co-immunoprecipitation with ATG7 antibodies to identify novel binding partners

  • Validate interactions using GST-pull-down assays as demonstrated with ATG7-PDCD4 interaction

  • Use proximity ligation assays to confirm interactions in intact cells

• Domain-specific mutants:

  • Generate constructs expressing mutant forms of ATG7 lacking autophagy-related domains

  • Use ATG7 antibodies to immunoprecipitate these mutants and identify retained interactions

  • Compare phenotypes between autophagy-deficient ATG7 mutants and wild-type ATG7

• Cellular localization studies:

  • Use subcellular fractionation followed by Western blotting with ATG7 antibodies

  • Perform immunofluorescence to track ATG7 localization during specific cellular processes

  • Investigate nuclear localization during transcriptional regulation events

Research has demonstrated that ATG7 regulates PDCD4 through proteasomal degradation in an autophagy-independent manner, evidenced by persistent regulation despite autophagy inhibition with Bafilomycin A1 and CQ .

What methodologies effectively distinguish between ATG7's roles in autophagy initiation versus elongation?

To differentiate ATG7's functions in autophagy initiation versus elongation:

• Temporal analysis:

  • Use time-course experiments with synchronized autophagy induction

  • Monitor ATG7 localization and activity at different time points using immunofluorescence

  • Correlate with early (initiation) and late (elongation) autophagy markers

• Structure-function studies:

  • Use domain-specific antibodies targeting different regions of ATG7

  • Create domain-specific ATG7 mutants and analyze effects on different autophagy stages

  • Monitor ATG7-mediated conjugation of ATG12 to ATG5 (initiation) versus LC3 lipidation (elongation)

• Interaction mapping:

  • Use immunoprecipitation with ATG7 antibodies at different autophagy stages

  • Identify stage-specific interaction partners

  • Perform proximity ligation assays to visualize spatiotemporal interactions

• Functional readouts:

  • Monitor ATG5-ATG12 conjugate formation (initiation phase)

  • Track LC3-I to LC3-II conversion (elongation phase)

  • Quantify autophagosome formation versus maturation using appropriate markers

This approach is supported by evidence showing that increasing doses of AdATG7 result in dose-dependent increases in autophagic flux, affecting both initiation and elongation phases of autophagy .

How does ATG7 modulate innate immune responses, and how can this be studied using ATG7 antibodies?

ATG7's role in innate immunity can be investigated through several methodological approaches:

• Viral infection models:

  • Compare viral replication in ATG7-overexpressing, wild-type, and ATG7-depleted cells

  • Use ATG7 antibodies to confirm expression levels via Western blotting and immunofluorescence

  • Recent research demonstrates that ATG7 overexpression facilitates viral replication while depletion attenuates it and renders mice more resistant to infection

• Interferon response analysis:

  • Measure type I and III interferon production in ATG7-manipulated cells during infection

  • Perform ChIP assays using ATG7 antibodies to identify potential transcriptional regulation

  • Evidence shows ATG7 restrains interferon production, with ATG7 depletion enhancing expression of type I and III interferons

• IRF3 activation studies:

  • Monitor IRF3 phosphorylation and nuclear translocation in relation to ATG7 levels

  • Use co-immunoprecipitation with ATG7 antibodies to identify interactions with IRF3 pathway components

  • Research demonstrates ATG7 significantly suppresses IRF3 activation during viral infection

• lncRNA regulatory mechanisms:

  • Investigate ATG7's control over lncRNA GAPLINC expression using RNA immunoprecipitation

  • Study how GAPLINC mediates the effects of ATG7 on innate immune responses

  • The ATG7/GAPLINC/IRF3 axis plays a critical role in regulating antiviral responses

• Animal model validation:

  • Use ATG7 conditional knockout mice to confirm in vitro findings

  • Perform immunohistochemistry with ATG7 antibodies to assess tissue-specific effects

  • ATG7 conditional knockout mice exhibit significant resistance to viral infections, with lower tissue injury and better survival compared to wild-type animals

What are common pitfalls when using ATG7 antibodies, and how can they be addressed?

Researchers frequently encounter several challenges when working with ATG7 antibodies:

• Background signal issues:

  • Problem: High background in immunoblotting or immunostaining

  • Solution: Optimize antibody concentration (start with 2 μg/mL for Western blot ), increase blocking time, use alternative blocking agents, and implement more stringent washing protocols

• Inconsistent detection in different cell types:

  • Problem: Variable ATG7 detection across cell lines

  • Solution: Validate antibody in each new cell type, adjust lysis conditions for different tissues, and optimize protein extraction for hard-to-lyse samples

• Species cross-reactivity concerns:

  • Problem: Unexpected or absent signals when working across species

  • Solution: Verify antibody species reactivity (human/mouse cross-reactivity has been confirmed ), use species-specific positive controls, and validate with knockout models

• Autophagic state influence:

  • Problem: ATG7 detection varies with autophagy status

  • Solution: Standardize sample collection timing, control autophagy induction conditions, and compare results using multiple antibodies targeting different ATG7 epitopes

• Fixation artifacts:

  • Problem: Altered staining patterns with different fixation methods

  • Solution: Optimize fixation protocols (4% paraformaldehyde is recommended for immunocytochemistry ), validate with multiple fixation approaches, and use heat-induced epitope retrieval for FFPE samples

How can researchers optimize co-immunoprecipitation protocols using ATG7 antibodies?

Successful co-immunoprecipitation with ATG7 antibodies requires careful optimization:

• Lysis buffer selection:

  • Use mild, non-denaturing buffers (e.g., NP-40 or CHAPS-based)

  • Include protease and phosphatase inhibitors to preserve interactions

  • Optimize salt concentration (150-300 mM NaCl range) to balance specificity and interaction strength

• Antibody coupling strategy:

  • Direct coupling to beads prevents heavy chain interference

  • For transient interactions, consider crosslinking approaches

  • Use appropriate controls including isotype antibodies and pre-clearing steps

• Interaction-specific considerations:

  • For ATG7-PDCD4 interaction: Cell lysis in NP-40 buffer followed by overnight incubation with antibodies has been validated

  • ATP-sensitive interactions: Maintain physiological ATP levels during extraction or manipulate ATP to study energy-dependent interactions

  • For detecting interactions under stress: Compare standard versus starvation conditions, as some interactions (e.g., ATG7-PDCD4) are attenuated during starvation

• Detection optimization:

  • For weak interactions: Increase protein input and reduce wash stringency

  • For specific interactions: Validate with reciprocal IP and confirmatory techniques like GST-pull-down

  • Consider native versus reducing conditions for Western blot detection of co-immunoprecipitated proteins

How should researchers interpret changes in ATG7 levels in relation to other autophagy markers?

Integrating ATG7 data with other autophagy markers requires careful interpretation:

How can ATG7 antibodies be utilized to study the therapeutic potential of ATG7 modulation in disease models?

ATG7 antibodies enable critical research into therapeutic applications across multiple disease contexts:

• Neurodegenerative disease models:

  • Track ATG7 expression and localization in affected neurons using immunohistochemistry

  • Monitor changes in autophagy flux following therapeutic interventions

  • Validate disease-modifying effects of ATG7 modulation through protein aggregation analysis

• Infection and immunity:

  • Use ATG7 antibodies to monitor expression during pathogen challenge and therapeutic intervention

  • Study the ATG7/GAPLINC/IRF3 axis as a potential therapeutic target

  • Research indicates that targeting ATG7 could enhance antiviral immunity, as ATG7 depletion renders mice more resistant to viral infections

• Cancer therapy development:

  • Employ ATG7 antibodies in immunohistochemistry to stratify tumors based on expression levels

  • Monitor autophagy dependency of tumors following treatment with autophagy modulators

  • Develop combination strategies targeting both ATG7-dependent autophagy and parallel survival pathways

• Metabolic disease interventions:

  • Track ATG7-PDCD4 interaction as a biomarker for metabolic stress response

  • Monitor ATP-sensing functions of ATG7 during metabolic interventions

  • Evidence shows ATG7 senses ATP levels and regulates AKT1-PDCD4 signaling during metabolic stress

• Therapeutic validation methodologies:

  • Implement tissue microarray analysis with ATG7 antibodies to determine expression across patient samples

  • Develop flow cytometry protocols to measure intracellular ATG7 levels as biomarkers

  • Use ATG7 antibodies in high-content imaging to screen for small molecule modulators of autophagy

What emerging techniques are enhancing the application of ATG7 antibodies in research?

Cutting-edge methodologies are expanding the utility of ATG7 antibodies:

• Advanced imaging applications:

  • Super-resolution microscopy: Visualize ATG7 localization at the nanoscale level during autophagosome formation

  • Live-cell imaging: Track ATG7 dynamics during autophagy using compatible antibody formats

  • Correlative light and electron microscopy (CLEM): Precisely localize ATG7 within ultrastructural contexts

• Single-cell technologies:

  • Mass cytometry (CyTOF): Multiplex ATG7 with dozens of other markers for comprehensive cellular profiling

  • Single-cell Western blotting: Analyze ATG7 expression heterogeneity within populations

  • Imaging mass cytometry: Spatially resolve ATG7 expression in tissue contexts with subcellular resolution

• Proximity labeling approaches:

  • BioID or APEX2 fusions with ATG7: Map the local interactome in different cellular compartments

  • Validation of proximity labeling results using co-immunoprecipitation with ATG7 antibodies

  • Identification of condition-specific interaction partners under different stresses

• Automated and high-throughput applications:

  • Simple Western systems: Automated capillary-based detection of ATG7 has been validated

  • High-content screening: Use ATG7 antibodies in large-scale functional genomics or drug screens

  • Microfluidic antibody-based assays: Develop sensitive detection methods for ATG7 in limited samples

These emerging techniques complement traditional applications like Western blotting, immunohistochemistry, and flow cytometry, expanding the research potential of ATG7 antibodies in both basic science and translational studies.