ATG5 Monoclonal Antibody

What is ATG5 and what is its role in autophagy?

ATG5 (Autophagy Related 5) is a 32 kDa protein that plays a crucial role in autophagosome formation and the broader autophagy pathway. It exists primarily as a covalent heterodimer with ATG12 through a specific Lys-Gly linkage. This ATG5-ATG12 conjugate functions as an E3-like enzyme required for the lipidation of ATG8 family proteins and their association with vesicle membranes, a process essential for autophagosome formation . ATG5 is ubiquitously expressed and contains N- and C-terminal ubiquitin-like domains (amino acids 15-105 and 187-275) separated by a helix-rich linker region with a critical lysine at position 130 for dimerization . The protein is essential for cellular homeostasis and survival during stress conditions such as nutrient deprivation .

What species reactivity do common ATG5 monoclonal antibodies show?

Most commercially available ATG5 monoclonal antibodies demonstrate cross-reactivity across multiple mammalian species. Based on validated experimental data, several monoclonal antibodies effectively detect ATG5 protein from human, mouse, and rat origins . Western blot analysis has confirmed detection in human cell lines (such as HeLa cervical epithelial carcinoma), mouse cell lines (including CH-1 B cell lymphoma), and rat cell lines (like PC-12 adrenal pheochromocytoma) . The high degree of sequence homology between species facilitates this cross-reactivity, as human ATG5 shares 97% amino acid identity with mouse ATG5 over amino acids 99-193 . When selecting an antibody for your research, it's advisable to verify the specific cross-reactivity data for your species of interest through the manufacturer's validation studies.

What techniques can ATG5 monoclonal antibodies be used for?

ATG5 monoclonal antibodies have been successfully validated for multiple research applications:

Researchers should note that these antibodies are available in various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, providing flexibility in experimental design .

How can I validate the specificity of an ATG5 monoclonal antibody?

Validating antibody specificity is crucial for reliable research outcomes. For ATG5 monoclonal antibodies, consider implementing these rigorous validation strategies:

• Positive and negative controls: Compare wild-type cells with ATG5 knockout cells or use siRNA-mediated knockdown of ATG5. The absence or significant reduction of signal in knockout/knockdown samples confirms specificity .

• Recombinant protein controls: Use purified recombinant ATG5 as a positive control. Testing with mutant forms like ATG5 K128A (which cannot conjugate with ATG12) can provide additional validation of recognition specificity .

• Molecular weight verification: True ATG5 antibodies should detect a band at approximately 50-55 kDa (free ATG5) and potentially a 70 kDa band representing the ATG5-ATG12 conjugate in Western blotting .

• Block with immunizing peptide: Pre-incubating the antibody with the peptide used for immunization should abolish specific signal.

• Multiple antibody comparison: Use two or more antibodies targeting different epitopes of ATG5 to confirm consistent detection patterns.

• Cross-species reactivity: If your antibody is claimed to recognize ATG5 from multiple species, verify detection in each species of interest using appropriate control samples .

These validation steps will ensure that experimental findings using ATG5 antibodies are reliable and reproducible.

What are the optimal conditions for Western blotting with ATG5 monoclonal antibodies?

For successful Western blot detection of ATG5, follow these optimized protocol recommendations:

• Sample preparation: Lyse cells in a buffer containing protease inhibitors to prevent degradation of ATG5. The ATG5-ATG12 conjugate can be sensitive to certain lysis conditions.

• Protein loading: Load 20-30 μg of total protein per lane for cell lysates. For tissue samples, 40-50 μg may be needed for clear detection.

• Gel separation: Use reducing conditions and 10-12% polyacrylamide gels for optimal separation of the 50-55 kDa (free ATG5) and 70 kDa (ATG5-ATG12 conjugate) bands .

• Transfer conditions: Transfer to PVDF membrane is recommended, as shown in validation studies with HeLa, CH-1, and PC-12 cell lines .

• Antibody concentration: Optimal working dilution ranges from 0.5-2 μg/mL depending on the specific antibody and sample type. For example, 0.5 μg/mL has been validated for detection in HeLa, CH-1, and PC-12 cell lysates .

• Detection system: HRP-conjugated secondary antibodies with ECL detection systems work well, but fluorescent-based detection systems may provide better quantitative results .

• Buffer system: Using appropriate immunoblot buffer systems is critical; Immunoblot Buffer Group 2 has been validated for certain ATG5 monoclonal antibodies .

Following these optimized conditions will help ensure consistent and specific detection of ATG5 in your Western blotting experiments.

What protocol should I use for immunohistochemical detection of ATG5?

For optimal immunohistochemical detection of ATG5 in tissue sections, follow this methodological approach:

• Fixation and embedding: Formalin-fixed, paraffin-embedded (FFPE) tissue sections yield optimal results for most ATG5 antibodies .

• Antigen retrieval: This critical step should involve heating tissue sections in 10 mM Tris with 1 mM EDTA, pH 9.0, for 45 minutes at 95°C, followed by cooling at room temperature for 20 minutes . This step is essential for unmasking ATG5 epitopes potentially masked during fixation.

• Blocking and antibody incubation: After antigen retrieval, block with appropriate serum (typically 5-10% normal serum from the same species as the secondary antibody). Incubate with the primary ATG5 monoclonal antibody for 30 minutes at room temperature or overnight at 4°C .

• Detection system: Use a detection system appropriate for the host species of your primary antibody. For mouse monoclonal antibodies, an anti-mouse detection system is suitable .

• Counterstaining and mounting: Counterstain with hematoxylin for nuclear visualization and mount with a compatible mounting medium.

• Controls: Always include positive control tissues known to express ATG5 and negative controls (omitting primary antibody or using isotype control antibodies) .

This protocol has been validated for mouse monoclonal ATG5 antibodies and should provide specific detection of ATG5 in tissue sections when properly optimized for your specific tissue type and antibody.

How does the ATG5-ATG12 conjugation system work, and how can I study it experimentally?

The ATG5-ATG12 conjugation system is a fundamental process in autophagy that resembles the ubiquitin conjugation pathway. To understand and study this system:

Biochemical mechanism: ATG12 is conjugated to ATG5 through a ubiquitin-like conjugating system that involves ATG7 as an E1-like activating enzyme and ATG10 as an E2-like conjugating enzyme . This conjugation forms a covalent bond between the C-terminal glycine (Gly185) of ATG12 and a specific lysine (Lys128) in ATG5 . The resulting ATG5-ATG12 conjugate then non-covalently associates with ATG16L1 multimers to form a complex that functions as an E3-like enzyme, facilitating the lipidation of ATG8 family proteins and their association with autophagosomal membranes .

Experimental approaches to study this system:

• In vitro reconstitution assay: This powerful approach allows you to study the conjugation process directly. Recombinant proteins (ATG5, ATG7, ATG10, and ATG12g - with exposed glycine) can be mixed with ATP to observe conjugate formation . Western blot analysis can detect the 50 kDa ATG5 and the 70 kDa ATG5-ATG12 conjugate . Control experiments should include:

  • Omitting ATP (negative control)

  • Omitting ATG7 or ATG10 (negative controls)

  • Substituting ATG3 for ATG10 (specificity control)

  • Using mutant ATG5 K128A (which cannot be conjugated)

• Cellular studies using mutants: Express mutant forms of ATG5 (K128A) or truncated ATG12 lacking the terminal glycine to disrupt conjugation and study functional consequences .

• Fluorescent tagging and microscopy: Use fluorescently tagged ATG5 and ATG12 to visualize the formation and localization of the conjugate during autophagy induction.

These approaches allow for detailed investigation of the conjugation mechanism and its role in autophagy and other cellular processes.

What are the non-canonical roles of ATG5 beyond autophagy?

ATG5 has emerged as a multifunctional protein with several important roles beyond canonical autophagy:

• Apoptosis regulation: ATG5 may play an important role in the apoptotic process, possibly through interaction with the modified cytoskeleton. Its expression appears to be a relatively late event in apoptosis, occurring downstream of caspase activity . Additionally, ATG5 plays a crucial role in IFN-gamma-induced autophagic cell death by interacting with FADD (Fas-Associated protein with Death Domain) .

• Immune system modulation: ATG5 is critical for multiple aspects of lymphocyte development and essential for both B and T lymphocyte survival and proliferation. It's also required for optimal processing and presentation of antigens for MHC II . This indicates ATG5's importance in adaptive immunity beyond simple autophagic functions.

• Lysosomal membrane protection: Recent research has revealed that ATG5, independent of other canonical autophagy factors, is necessary for lysosomes to recruit ALIX and ESCRT repair machinery . In ATG5 knockout cells, lysosomes are more vulnerable to damage, which contributes to a range of exocytic and secretory phenotypes .

• Viral infection response: ATG5, in association with ATG12, can negatively regulate the innate antiviral immune response by impairing type I IFN production upon vesicular stomatitis virus (VSV) infection . This suggests a proviral function in certain contexts.

• Cellular structure maintenance: ATG5 is involved in the maintenance of axon morphology and membrane structures, as well as in normal adipocyte differentiation .

• Primary ciliogenesis: ATG5 promotes primary ciliogenesis through the removal of OFD1 from centriolar satellites and degradation of IFT20 via the autophagic pathway .

These non-canonical functions highlight the importance of studying ATG5 beyond its role in canonical autophagy, particularly in contexts such as immunity, cell death, and cellular homeostasis.

How can I differentiate between canonical and non-canonical functions of ATG5 in my research?

Differentiating between canonical autophagy-dependent and autophagy-independent functions of ATG5 requires careful experimental design:

• Comparative genetic approaches: Compare phenotypes between ATG5 knockout cells and knockouts of other essential autophagy genes (e.g., ATG7, ATG3, ATG16L1). Functions that are disrupted only in ATG5 knockouts but not in other autophagy gene knockouts likely represent non-canonical roles . For example, studies show that knockout of ATG5 in myeloid cells during Mycobacterium tuberculosis infection caused neutrophilic inflammation and increased mortality, while loss of other ATG genes had no apparent consequences in short-term studies .

• Rescue experiments with mutants: Use structure-function analysis by expressing:

  • Wild-type ATG5

  • ATG5 K130A (unable to conjugate with ATG12, disrupting canonical autophagy)

  • Other domain-specific mutants

  Functions rescued by mutants defective in autophagy likely represent non-canonical roles.

• Lysosomal function assays: Since ATG5 has unique roles in lysosomal membrane protection independent of canonical autophagy, measure lysosomal integrity, recruitment of ALIX and ESCRT machinery, and membrane repair processes in ATG5-deficient versus autophagy-deficient (other ATG knockouts) cells .

• Biochemical isolation of protein complexes: Use co-immunoprecipitation and mass spectrometry to identify ATG5-interacting proteins. Interactions unique to ATG5 (not shared with ATG12-ATG5 conjugate) may indicate non-canonical functions.

• Selective autophagy receptor analysis: Determine if phenotypes are dependent on specific autophagy receptors (p62/SQSTM1, NBR1, etc.) or independent of these receptors, which would suggest non-canonical functions.

By systematically comparing ATG5 functions with canonical autophagy processes, researchers can distinguish between ATG5's roles in autophagy and its independent cellular functions.

Why might I detect multiple bands in Western blot using ATG5 antibodies?

Multiple bands in Western blots with ATG5 antibodies can have several scientific explanations:

• Expected multiple forms: The primary expected bands are:

  • Free ATG5: ~50-55 kDa band representing unconjugated ATG5

  • ATG5-ATG12 conjugate: ~70 kDa band representing the covalent conjugate formed between ATG5 and ATG12

• Alternative start sites: Human ATG5 has two potential alternative start sites at Met80 and Met173, which could generate truncated forms of the protein with lower molecular weights . These may appear as additional bands of approximately 22 kDa and 12 kDa, respectively.

• Post-translational modifications: ATG5 can undergo various modifications affecting its migration pattern, including:

  • Phosphorylation: Can result in slightly higher molecular weight bands

  • Proteolytic cleavage: During apoptosis, ATG5 can be cleaved, generating fragments

• Cross-reactivity: Some antibodies may cross-react with structurally similar proteins, particularly those with ubiquitin-like domains similar to ATG5's N- and C-terminal domains (aa 15-105 and 187-275) .

• Technical factors:

  • Incomplete reduction: Ensure complete reduction with fresh DTT or β-mercaptoethanol

  • Sample degradation: Use fresh samples with protease inhibitors

  • Non-specific binding: Optimize blocking conditions and antibody concentration

To distinguish between specific and non-specific bands, consider:

• Including lysates from ATG5 knockout cells as negative controls

• Using recombinant ATG5 as a positive control

• Testing with antibodies targeting different epitopes of ATG5

• Performing peptide competition assays

Understanding the origin of multiple bands is crucial for accurate interpretation of Western blot results in ATG5 research.

How should I interpret changes in ATG5 levels during autophagy induction?

Interpreting changes in ATG5 levels during autophagy induction requires careful consideration of several factors:

• Free ATG5 vs. ATG5-ATG12 conjugate: The ratio between free ATG5 (~50-55 kDa) and the ATG5-ATG12 conjugate (~70 kDa) is more informative than total ATG5 levels. During autophagy induction, you may observe:

  • Increased ATG5-ATG12 conjugate formation

  • Decreased free ATG5 levels

  • Relatively stable total ATG5 expression

• Time-course considerations: ATG5's role in autophagy is primarily functional rather than being consumed in the process. Therefore:

  • Early after autophagy induction (1-4 hours): Minimal changes in total ATG5 levels are expected

  • Extended autophagy (>12 hours): Potential upregulation of ATG5 expression to sustain autophagy

  • Recovery phase: Return to baseline levels

• Cell type-specific regulation: Different cell types exhibit varying baseline levels and regulation patterns of ATG5 during autophagy induction:

  • Immune cells may show more dynamic regulation

  • Neurons and cardiac cells often maintain more stable ATG5 levels

• Autophagy flux vs. ATG5 levels: Changes in LC3-II levels or p62/SQSTM1 degradation more directly indicate autophagy flux, while ATG5 levels reflect the capacity for autophagy rather than the current rate.

• Experimental controls for interpretation:

  • Include positive controls (starvation, rapamycin treatment)

  • Use autophagy inhibitors (bafilomycin A1, chloroquine) to determine flux

  • Compare ATG5 expression with other autophagy proteins (ATG7, ATG16L1)

When reporting changes in ATG5 levels, always:

• Quantify band intensities with appropriate normalization

• Present both free ATG5 and ATG5-ATG12 conjugate data

• Correlate with functional autophagy assays

• Consider cell type-specific and stimulus-specific contexts

These principles will help ensure accurate interpretation of ATG5 dynamics during autophagy research.

What experimental controls should I include when studying ATG5 in autophagy research?

Robust experimental controls are essential when studying ATG5 in autophagy research:

• Genetic controls:

  • ATG5 knockout/knockdown cells: Essential negative controls that should show absence of ATG5 protein and impaired autophagosome formation

  • ATG5 reconstitution: Rescue experiments expressing wild-type ATG5 in knockout cells validate phenotype specificity

  • K128A mutant ATG5: This mutant cannot conjugate with ATG12, providing a functional separation of ATG5's roles

• Autophagy pathway controls:

  • Other ATG gene knockouts: Compare with ATG7, ATG12, or ATG16L1 knockouts to distinguish between shared pathway effects vs. ATG5-specific functions

  • Upstream regulators: Manipulate mTOR, AMPK, or ULK1 to verify canonical autophagy pathway engagement

  • Downstream effects: Monitor LC3 lipidation and p62/SQSTM1 degradation as functional readouts

• Pharmacological controls:

  • Autophagy inducers: Rapamycin, Torin1, or starvation as positive controls

  • Autophagy inhibitors: Bafilomycin A1 (blocks autophagosome-lysosome fusion) or chloroquine (disrupts lysosomal function)

  • Lysosomal inhibitors: Separate autophagy induction from degradation phases

• Technical controls for antibody-based detection:

  • Loading controls: β-actin, GAPDH, or tubulin for normalization

  • Recombinant protein: Purified ATG5 or ATG5-ATG12 conjugate as positive controls

  • Cross-reactivity controls: Test antibodies across species when working with non-human models

  • Blocking peptides: Confirm antibody specificity

• Experimental design controls:

  • Time-course analysis: Capture the dynamic nature of autophagy

  • Multiple detection methods: Combine Western blot, IF, and functional assays

  • Cell density standardization: Control for contact inhibition effects on autophagy

Including these controls will strengthen the validity of your ATG5 research findings and help distinguish between canonical autophagy functions and non-canonical roles of ATG5.

What are the latest findings on ATG5's role in immune responses and disease?

Recent research has revealed critical roles for ATG5 in immune regulation and disease processes:

• Infection response mechanisms: ATG5 provides host protection by acting as a molecular switch in the atg8ylation pathway during infection . In murine models of Mycobacterium tuberculosis (Mtb) infection, genetic knockout of ATG5 specifically in myeloid cells resulted in severe neutrophilic inflammation and increased mortality, while knockout of other autophagy genes showed minimal effects in short-term studies . This suggests ATG5 has unique immunoprotective functions beyond the canonical autophagy pathway.

• Lysosomal membrane integrity: ATG5 has been discovered to have a unique role in maintaining lysosomal membrane integrity. In the absence of ATG5 (but not other autophagy factors), lysosomes cannot recruit ALIX and ESCRT repair machinery, making them more vulnerable to damage . This vulnerability contributes to various exocytic and secretory phenotypes that can influence immune responses.

• Dual role in antiviral immunity: While ATG5 can facilitate xenophagy (autophagy of pathogens), it also plays a complex regulatory role in antiviral responses. In association with ATG12, ATG5 can negatively regulate innate antiviral immune responses by impairing type I interferon production during vesicular stomatitis virus (VSV) infection . This suggests a context-dependent role that may be exploited by certain pathogens.

• Lymphocyte development and function: ATG5 plays critical roles in multiple aspects of lymphocyte development and is essential for both B and T lymphocyte survival and proliferation . It's also required for optimal processing and presentation of antigens for MHC II, linking autophagy machinery to adaptive immune functions .

• Parasitic infection response: Studies with Leishmania have demonstrated that ATG5 is essential for ATG8-dependent autophagy and the cellular remodeling required for parasite differentiation . This highlights ATG5's involvement in host-parasite interactions.

These findings emphasize the need to consider ATG5's multifaceted roles beyond canonical autophagy when interpreting immune phenotypes and developing therapeutic strategies targeting autophagy in infectious and inflammatory diseases.

How can I use ATG5 as a marker or target in autophagy-related disease research?

ATG5 serves as a valuable marker and potential therapeutic target in autophagy-related disease research:

• As a diagnostic/prognostic marker:

  • Cancer: Altered ATG5 expression has been observed in various cancers. Quantifying the ATG5-ATG12 conjugate versus free ATG5 ratio may provide insights into autophagy status in tumor samples. Use immunohistochemistry with validated ATG5 monoclonal antibodies on tissue microarrays for systematic analysis .

  • Neurodegenerative diseases: ATG5 levels or its subcellular distribution may reflect impaired autophagy in conditions like Alzheimer's, Parkinson's, and Huntington's diseases. Combine ATG5 detection with markers of protein aggregation (β-amyloid, α-synuclein, huntingtin) in tissue sections or cellular models .

  • Inflammatory conditions: Monitor ATG5 expression in inflammatory cells, particularly in conditions where autophagy dysfunction contributes to pathogenesis, such as inflammatory bowel disease .

• As an experimental tool:

  • Cell-specific ATG5 knockout models: Generate tissue-specific ATG5 knockout animals to study autophagy's role in specific physiological contexts. This approach has revealed critical roles in myeloid cells during Mycobacterium tuberculosis infection .

  • ATG5 reconstitution systems: Use ATG5-deficient cells reconstituted with wild-type or mutant ATG5 (e.g., K128A that cannot conjugate with ATG12) to dissect autophagy-dependent versus independent functions .

  • ATG5 reporter systems: Develop fluorescent ATG5 fusion proteins to monitor autophagy dynamics in real-time in disease models.

• As a therapeutic target:

  • Structure-based drug design: Target the ATG5-ATG12 interaction interface for developing small molecule modulators of autophagy. The conjugation system involving ATG7 (E1-like), ATG10 (E2-like), and ATG5-ATG12 (E3-like) offers multiple potential intervention points .

  • Context-specific modulation: Develop approaches to selectively enhance or inhibit ATG5's functions in particular cellular contexts, such as boosting ATG5 activity in neurodegenerative conditions while inhibiting it in certain cancers where autophagy promotes tumor cell survival.

• For mechanistic disease understanding:

  • Interaction proteomics: Identify disease-specific ATG5 interaction partners using immunoprecipitation with validated ATG5 antibodies followed by mass spectrometry .

  • Post-translational modification mapping: Characterize how disease states affect ATG5 modifications and function using site-specific antibodies or mass spectrometry approaches.

By utilizing ATG5 as both a marker and target, researchers can gain deeper insights into autophagy's role in disease pathogenesis and develop novel therapeutic strategies.