ATG3 Antibody

What is ATG3 and why is it important in autophagy research?

ATG3 is an E2 conjugating enzyme that catalyzes the covalent conjugation of ATG8-like proteins (including GABARAP, GABARAPL1, GABARAPL2, and MAP1LC3A) to phosphatidylethanolamine (PE)-containing lipids in cellular membranes. This conjugation is one of the critical steps in autophagosome formation during autophagy. ATG3 functions by cycling between ATG7 (which loads it with ATG8-like proteins) and the E3 enzyme complex (composed of ATG12, ATG5, and ATG16L1), which promotes the lipidation of ATG8-like proteins . Beyond its role in conventional autophagy, ATG3 also functions as a membrane curvature sensor that facilitates LC3/GABARAP lipidation by detecting local membrane stress associated with lipid-packing defects. Additionally, ATG3 contributes to mitochondrial homeostasis, endosome-lysosome trafficking, and primary ciliogenesis . Studying ATG3 using specific antibodies provides crucial insights into autophagy regulation and related cellular processes.

What types of ATG3 antibodies are available for research?

There are several types of ATG3 antibodies available for research applications, with the most common being:

• Mouse monoclonal antibodies - such as clone OTI3C6 (PrecisionAb), which is an IgG2a isotype antibody purified by affinity chromatography from ascites .

• Rabbit recombinant monoclonal antibodies - such as clone EPR4801, which has been validated for multiple applications and cited in numerous scientific publications .

The choice between these antibody types depends on your specific experimental needs, including the detection method, target species, and application requirements. Mouse monoclonal antibodies often provide high specificity and low background, while rabbit monoclonal antibodies frequently offer high sensitivity and work well in multiple applications including immunohistochemistry and Western blotting .

What is the molecular weight of ATG3 and how is this relevant for antibody validation?

ATG3 has a predicted molecular weight of approximately 35 kDa, but it consistently appears at approximately 40 kDa in Western blot analyses . This discrepancy between the predicted and observed molecular weights is an important consideration when validating ATG3 antibodies. When performing Western blot analysis, researchers should expect to observe a band at approximately 40 kDa, as consistently reported in the literature and antibody validation data. For example, both Bio-Rad's mouse anti-ATG3 (clone OTI3C6) and Abcam's rabbit anti-ATG3 (clone EPR4801) detect ATG3 at approximately 40 kDa in various cell lysates including Jurkat, HeLa, K562, and HL-60 cells . This information is crucial for proper interpretation of Western blot results and confirmation of antibody specificity.

How should I validate an ATG3 antibody before using it in my experiments?

Thorough validation of ATG3 antibodies is essential for generating reliable and reproducible results. A comprehensive validation approach should include:

• Western blot analysis using positive control lysates (e.g., Jurkat, HeLa, K562 cells) to confirm detection of the expected 40 kDa band .

• Negative controls using knockout cell lines, such as ATG3 knockout HEK293T cells, to confirm antibody specificity. The absence of signal in knockout samples strongly supports antibody specificity .

• Cross-reactivity testing if working with non-human samples. For example, some ATG3 antibodies show cross-reactivity with mouse and rat samples, though reactivity and working conditions may vary between species .

• Application-specific validation for techniques beyond Western blotting, such as immunohistochemistry, immunofluorescence, or flow cytometry. For instance, the EPR4801 clone has been validated for immunofluorescent staining of HeLa cells at 1/100 dilution and for intracellular flow cytometry at 1/2000 dilution .

• Literature review to identify previously validated antibodies and protocols. Antibodies cited in multiple publications generally have more reliable validation data.

Proper validation not only ensures experimental success but also prevents wasted time and resources on suboptimal or non-specific antibodies.

What are the optimal conditions for using ATG3 antibodies in Western blotting?

For optimal Western blotting results with ATG3 antibodies, consider the following methodological approaches:

• Sample preparation: Prepare cell lysates from autophagy-relevant cell lines such as HeLa, Jurkat, or K562. Typically, 10-20 μg of total protein per lane is sufficient for detection .

• Antibody dilutions:

  • For mouse monoclonal antibodies like OTI3C6: Use at approximately 0.5 mg/ml concentration

  • For rabbit monoclonal antibodies like EPR4801: Optimal dilutions range from 1/1000 to 1/10000 depending on the detection system and sample type

• Detection systems: Both HRP-conjugated secondary antibodies and fluorescently-labeled secondaries (e.g., IRDye® 800CW or 680RD) provide excellent results. Fluorescent detection offers advantages for quantification and multiplexing .

• Expected results: Look for a specific band at approximately 40 kDa. This is consistent across various cell types despite the predicted molecular weight of 35 kDa .

• Controls: Always include positive controls (known ATG3-expressing cells) and, when possible, negative controls such as ATG3 knockout cell lysates to confirm specificity .

• Buffer conditions: Standard Western blot protocols with PBS-based buffers work well, though specific buffer optimizations may be required for challenging samples or to reduce background .

Following these guidelines will help ensure consistent and reliable detection of ATG3 in Western blotting applications.

How can I effectively use ATG3 antibodies to study autophagy induction and flux?

To effectively study autophagy induction and flux using ATG3 antibodies, implement the following methodological approaches:

• Experimental design: Pair ATG3 detection with key autophagy markers, particularly LC3-I to LC3-II conversion, since ATG3 directly catalyzes LC3 lipidation. This combination provides more comprehensive insights into autophagy regulation .

• Starvation protocols: Induce autophagy through nutrient deprivation (e.g., using Hanks' balanced salt solution) for 90-120 minutes. Adding chloroquine (20 μM) prevents autophagosome degradation, allowing for accumulation and easier detection of autophagy-related proteins .

• Inhibitor studies: Compare samples with and without autophagy inhibitors (e.g., 3-methyladenine) or lysosomal inhibitors (e.g., bafilomycin A1) to differentiate between altered autophagy induction versus flux.

• Quantification: Measure both ATG3 levels and LC3-II/LC3-I ratios. For accurate quantification, use infrared imaging systems (like LI-COR Odyssey) and appropriate analysis software (e.g., Image Studio) .

• Complementary approaches: Combine Western blotting with fluorescence microscopy using immunofluorescence to visualize the colocalization of ATG3 with autophagosomal markers.

• Genetic approaches: Use ATG3 mutants that affect its function (e.g., the D156A/M157A double mutant that impairs interaction with ATG12~ATG5) to analyze the relationship between ATG3 function and autophagy markers .

This integrated approach allows researchers to comprehensively assess how ATG3 contributes to autophagy induction, elongation, and flux under different experimental conditions.

What are the technical considerations for immunoprecipitation assays using ATG3 antibodies?

When performing immunoprecipitation (IP) assays with ATG3 antibodies, several technical considerations are essential for successful experiments:

• Antibody selection: Choose antibodies specifically validated for IP applications. Not all Western blot-validated antibodies work efficiently for IP .

• Cell lysis conditions: Use mild lysis buffers that preserve protein-protein interactions. Harsh detergents may disrupt important ATG3 interactions with partners like ATG12~ATG5 or ATG7 .

• Antibody binding: For optimal results, incubate cell lysates with anti-ATG3 antibodies overnight at 4°C with gentle rotation to ensure sufficient binding while minimizing degradation .

• Pull-down methods: Protein A/G beads work well for rabbit antibodies, while Protein G beads are preferred for mouse antibodies. Pre-clearing lysates with beads alone can reduce non-specific binding .

• Co-immunoprecipitation targets: When studying ATG3 interactions, probe for known binding partners such as:

  • ATG12~ATG5 conjugate (approximately 55 kDa)

  • ATG7 (78 kDa)

  • LC3 (16 kDa for LC3-I; 14 kDa for LC3-II)

• Controls: Include negative controls (non-specific IgG or ATG3 knockout lysates) and input samples (pre-IP lysate) to assess IP efficiency and specificity .

• Mutant analysis: Consider using ATG3 mutants (e.g., D156A/M157A) that disrupt specific protein interactions to confirm the specificity of co-IP results. These mutants have been shown to abolish ATG12~ATG5 binding while maintaining ATG7 interaction .

Following these guidelines will help ensure successful immunoprecipitation experiments that provide insights into ATG3's protein interaction network during autophagy.

How do I optimize immunofluorescence protocols using ATG3 antibodies?

Optimizing immunofluorescence protocols with ATG3 antibodies requires attention to several key parameters:

Following these guidelines will help researchers obtain clear and specific immunofluorescence staining for ATG3, facilitating studies of its subcellular localization and potential redistribution during autophagy induction.

How can I use ATG3 antibodies to study the structural interactions between ATG3 and other autophagy proteins?

Studying structural interactions between ATG3 and its binding partners requires specialized approaches beyond basic antibody applications:

• Epitope mapping: Select antibodies that recognize distinct epitopes on ATG3, particularly those that do not interfere with protein-protein interaction domains. The region between residues 140-170 of ATG3 (RIA12) is crucial for interaction with ATG12 and should be considered when choosing antibodies for interaction studies .

• Competitive binding assays: Use purified recombinant ATG3 fragments (such as ATG3 FR consisting of residues 88-192) in combination with antibodies to assess whether antibody binding competes with binding of interaction partners like ATG12~ATG5 .

• Structural domain analysis: Design experiments that focus on specific ATG3 domains, particularly:

  • The flexible region (ATG3 FR) inserted between β2 and β3 of the E2 core, which is involved in both ATG7 and E3 binding

  • The RIA12 region (residues 140-170) that specifically interacts with ATG12

• Mutational analysis: Incorporate critical point mutations known to disrupt specific interactions, such as:

  • D156A and M157A: Severely impair ATG12 binding (increases Kᴅ by 18 to 330-fold)

  • Y160A and E161A: Significantly affect ATG12 binding

  • 8×Ala mutation (residues 144-151): Disrupts the cluster of negatively charged residues important for high-affinity interactions

• Co-immunoprecipitation with structure-specific antibodies: Use antibodies that recognize specific conformational states of ATG3 to isolate and identify interaction partners under different cellular conditions.

• Proximity ligation assays (PLA): Combine two antibodies (anti-ATG3 and antibodies against potential interaction partners) to visualize and quantify protein-protein interactions in situ with high sensitivity.

These approaches, combined with structural data from X-ray crystallography, provide valuable insights into how ATG3 structurally interacts with the autophagy machinery to facilitate LC3 lipidation and autophagosome formation.

What methods can I use to analyze ATG3-mediated LC3 lipidation efficiency in complex experimental models?

Analyzing ATG3-mediated LC3 lipidation efficiency in complex experimental models requires sophisticated approaches that go beyond simple Western blotting:

• LC3-II formation assays in genetically modified systems: Establish stable cell lines expressing wild-type and mutant ATG3 constructs (e.g., D156A, M157A, or D156A/M157A double mutants) in ATG3-knockout backgrounds. Monitor LC3-II formation under various autophagy-inducing conditions using Western blotting with anti-LC3 antibodies .

• Quantitative analysis: Use infrared imaging systems (such as LI-COR Odyssey) for precise quantification of LC3-II/LC3-I ratios and normalize to appropriate loading controls (e.g., α-tubulin). This approach allows direct correlation between ATG3 mutations and LC3 lipidation efficiency .

• Kinetic studies: Implement time-course experiments to assess the rate of LC3 lipidation rather than just endpoint measurements. Compare lipidation kinetics between wild-type and mutant ATG3 variants to identify rate-limiting steps.

• In vitro lipidation assays: Reconstitute the LC3 lipidation process using purified components (ATG7, ATG3, ATG12~ATG5-ATG16N, LC3, and synthetic liposomes) to directly measure the enzymatic efficiency of wild-type versus mutant ATG3 proteins.

• Structure-function analysis: Correlate lipidation efficiency with binding affinity measurements obtained through isothermal titration calorimetry (ITC). For example, the D156A/M157A double mutation both abolishes ATG12~ATG5 binding (as measured by ITC) and eliminates LC3-II formation in cells .

• Organelle-specific lipidation: Develop assays to distinguish LC3 lipidation occurring on different membrane compartments (autophagosomes, mitochondria, endosomes) using organelle fractionation combined with Western blotting or fluorescence microscopy.

• Live-cell imaging: Monitor LC3 lipidation dynamics in real-time using fluorescently tagged LC3 in cells expressing wild-type or mutant ATG3, allowing for assessment of both spatial and temporal aspects of the lipidation process.

These integrated approaches provide comprehensive insights into how ATG3 structure and interactions influence its lipidation activity in complex cellular environments.

How can I analyze the impact of post-translational modifications of ATG3 using specific antibodies?

Analyzing post-translational modifications (PTMs) of ATG3 requires specialized antibodies and methodological approaches:

• Phosphorylation-specific antibodies: Develop or obtain antibodies that specifically recognize phosphorylated residues of ATG3. While not mentioned in the provided search results, phosphorylation is a common regulatory mechanism for E2 enzymes.

• Fractionation approaches: Combine subcellular fractionation with Western blotting using anti-ATG3 antibodies to determine how PTMs affect the subcellular distribution of ATG3. For example, membrane association versus cytosolic localization may be influenced by specific modifications.

• Two-dimensional gel electrophoresis: Separate ATG3 based on both molecular weight and isoelectric point to resolve differentially modified forms, followed by Western blotting with anti-ATG3 antibodies.

• Immunoprecipitation and mass spectrometry: Use anti-ATG3 antibodies to immunoprecipitate the protein from cells under different conditions (basal, starvation, stress), followed by mass spectrometry analysis to identify and quantify specific PTMs.

• Modification-specific functional assays: Compare the activity of differently modified ATG3 forms in LC3 lipidation assays. For example, assess how phosphorylation affects:

  • Binding to ATG7 (E1)

  • Interaction with ATG12~ATG5 (E3)

  • Membrane association

  • LC3 conjugation efficiency

• PTM-mimicking mutants: Generate ATG3 mutants that either mimic or prevent specific modifications (e.g., phosphomimetic S/T→D/E or non-phosphorylatable S/T→A mutations) and assess their function in ATG3-knockout backgrounds.

• Inhibitor studies: Use specific kinase or other PTM enzyme inhibitors to manipulate ATG3 modification status, then analyze the consequences using anti-ATG3 antibodies in combination with functional autophagy assays.

This multi-faceted approach allows researchers to comprehensively characterize how post-translational modifications regulate ATG3 function in autophagy and related cellular processes.

What are the optimal approaches for studying ATG3's role in non-canonical autophagy pathways?

To investigate ATG3's role in non-canonical autophagy pathways, researchers should employ specialized methodological approaches:

• ATG3-ATG12 conjugate analysis: Study the autocatalytic conjugation of ATG12 to ATG3 (distinct from the conventional ATG5-ATG12 conjugation) using Western blotting with anti-ATG3 antibodies. This ATG3-ATG12 conjugate is implicated in mitochondrial homeostasis and endosome-lysosome trafficking rather than conventional autophagy .

• Differential pathway inhibition: Use selective inhibitors or genetic knockdowns to distinguish between canonical and non-canonical pathways:

  • Compare ATG3-dependent processes that are also dependent on ULK1 complex (canonical) versus those that are ULK1-independent (often non-canonical)

  • Analyze processes requiring ATG3 but not other core autophagy proteins

• Organelle-specific autophagy: Investigate ATG3's role in selective autophagy processes using co-localization studies with organelle-specific markers:

  • Mitophagy (mitochondrial markers)

  • Pexophagy (peroxisome markers)

  • ER-phagy (ER markers)

  • Xenophagy (pathogen markers)

• Membrane association studies: Analyze ATG3's membrane-sensing capabilities and interaction with negatively-charged membranes, which may be particularly relevant for non-canonical functions. This can be studied through in vitro membrane binding assays and cellular fractionation approaches .

• Conditional knockout models: Generate tissue-specific or inducible ATG3 knockout models to distinguish between developmental roles and acute functions in specific tissues or conditions.

• Interaction network analysis: Use immunoprecipitation with anti-ATG3 antibodies followed by mass spectrometry to identify novel interaction partners specific to non-canonical pathways. Pay particular attention to:

  • Endosomal proteins (given ATG3's role in endosome-lysosome trafficking)

  • Mitochondrial proteins

  • Proteins involved in primary ciliogenesis

• Live-cell imaging: Employ fluorescently tagged ATG3 combined with organelle markers to track its recruitment and function during non-canonical autophagy events in real-time.

These approaches will help delineate ATG3's diverse functions beyond conventional autophagy and provide insights into its roles in specialized cellular processes.

How can I address common issues with ATG3 antibody specificity and sensitivity?

When facing specificity or sensitivity issues with ATG3 antibodies, implement these troubleshooting strategies:

• Antibody validation using genetic controls:

  • Compare signal in wild-type versus ATG3 knockout cell lines (e.g., ATG3 knockout HEK293T cells)

  • If the antibody detects bands in knockout samples, it likely has cross-reactivity issues

• Epitope competition assays:

  • Pre-incubate the antibody with recombinant ATG3 protein or immunizing peptide

  • A reduction in signal indicates specific binding to the target epitope

• Sensitivity optimization for Western blotting:

  • Try different dilutions ranging from 1/1000 to 1/10000

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • For fluorescent detection systems, optimize scanner settings

• Background reduction strategies:

  • Increase blocking time and concentration (5% BSA or milk)

  • Add 0.1-0.3% Tween-20 to washing buffers

  • For tissues with high endogenous biotin, include an avidin/biotin blocking step

  • Use more stringent washing conditions (higher salt concentration, longer washes)

• Sample preparation optimization:

  • Ensure complete cell lysis to release all ATG3 protein

  • Include protease inhibitors to prevent degradation

  • Fresh samples generally yield better results than frozen-thawed samples

• Antibody storage and handling:

  • Avoid repeated freeze-thaw cycles

  • Store antibodies according to manufacturer recommendations (often with 50% glycerol at -20°C)

  • Centrifuge antibody vials before use to remove aggregates

• Alternative antibody evaluation:

  • Compare multiple antibodies targeting different epitopes of ATG3

  • Consider switching from monoclonal to polyclonal (or vice versa) depending on the application

These systematic troubleshooting approaches will help resolve most specificity and sensitivity issues encountered with ATG3 antibodies.

What strategies can I employ to optimize detection of ATG3 in different tissue types and species?

Optimizing ATG3 detection across different tissue types and species requires careful consideration of several factors:

• Species cross-reactivity assessment:

  • Review antibody documentation for validated cross-reactivity (e.g., some ATG3 antibodies cross-react with human, mouse, and rat samples)

  • Perform preliminary validation in your specific species using positive control samples

  • Be aware that antibody reactivity and optimal working conditions may vary significantly between species

• Tissue-specific optimization for immunohistochemistry:

  • Adjust fixation protocols based on tissue type (duration, fixative concentration)

  • Optimize antigen retrieval methods:

    • Heat-induced epitope retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

    • Enzymatic retrieval (proteinase K or trypsin)

  • Test different antibody concentrations and incubation times for each tissue type

• Sample preparation considerations:

  • For fibrous tissues (muscle, skin), extend protease digestion time

  • For tissues with high endogenous peroxidase (liver, kidney), increase H₂O₂ quenching time

  • For fatty tissues, ensure complete deparaffinization and consider lipid extraction steps

• Background reduction strategies for different tissues:

  • For tissues with high background, add 0.1-0.3% Triton X-100 to antibody diluent

  • For highly autofluorescent tissues, use Sudan Black B treatment or specialized quenching reagents

  • Consider mouse-on-mouse blocking reagents when using mouse antibodies on mouse tissues

• Detection system optimization:

  • For tissues with low ATG3 expression, use signal amplification systems (tyramide signal amplification, polymer-based detection)

  • For multiplexing applications, select antibody combinations raised in different host species

• Validation approaches:

  • Include tissue from ATG3 knockout animals as negative controls

  • Use tissues with known high ATG3 expression (e.g., liver, brain) as positive controls

  • Verify staining patterns with multiple antibodies targeting different epitopes

• Western blot considerations for tissue samples:

  • Optimize tissue homogenization and lysis buffer composition for each tissue type

  • Adjust protein loading (10-50 μg) based on ATG3 expression levels in different tissues

These tissue and species-specific optimizations will help ensure reliable ATG3 detection across diverse experimental systems.

How should I interpret conflicting ATG3 antibody results in autophagy research?

When faced with conflicting ATG3 antibody results in autophagy research, employ these analytical and troubleshooting approaches:

• Epitope considerations:

  • Different antibodies may recognize distinct epitopes on ATG3

  • Certain epitopes might be masked in protein complexes or affected by post-translational modifications

  • Map the epitopes of your antibodies and consider how structural changes during autophagy might affect epitope accessibility

• Methodological variables analysis:

  • Create a detailed comparison table of experimental conditions (fixation, permeabilization, blocking, antibody concentrations)

  • Systematically test each variable to identify which factors contribute to the discrepancies

  • Consider whether differences in sample preparation might explain conflicting results

• Biological context evaluation:

  • Different cell types or tissues may exhibit varying ATG3 expression levels or isoforms

  • Autophagy induction methods (starvation, rapamycin, etc.) might differentially affect ATG3 detection

  • Consider the timing of analysis, as ATG3 localization and modification state can change during autophagy progression

• Antibody quality assessment:

  • Evaluate lot-to-lot variation by testing multiple lots of the same antibody

  • Review validation data for each antibody, including knockout controls

  • Consider antibody age and storage conditions, as antibody quality can deteriorate over time

• Integration with complementary techniques:

  • Corroborate antibody-based results with non-antibody methods:

    • mRNA expression analysis (qPCR, RNA-seq)

    • Fluorescently tagged ATG3 constructs

    • Mass spectrometry-based protein quantification

  • Use genetic approaches (siRNA knockdown, CRISPR knockout) to validate antibody specificity

• Expert consultation and literature review:

  • Contact antibody manufacturers regarding known issues or limitations

  • Review literature for similar conflicts and how they were resolved

  • Consult with laboratories experienced in ATG3 research for technical insights

• Decision framework for data interpretation:

  • Weight results based on validation strength (antibodies validated with knockout controls should be given higher confidence)

  • Consider whether conflicting results might represent real biological phenomena rather than technical artifacts

  • Be transparent about conflicts in publications and present multiple lines of evidence

What innovative techniques can be combined with ATG3 antibodies for advanced autophagy research?

Combining ATG3 antibodies with innovative techniques can significantly advance autophagy research:

• Super-resolution microscopy approaches:

  • Stimulated emission depletion (STED) microscopy to visualize ATG3 localization with nanometer precision

  • Stochastic optical reconstruction microscopy (STORM) to map ATG3 distribution relative to autophagosomal membranes

  • Structured illumination microscopy (SIM) for improved resolution of ATG3 dynamics during autophagosome formation

• Proximity labeling techniques:

  • BioID or TurboID fused to ATG3 to identify proximal proteins in living cells

  • APEX2-ATG3 fusion for electron microscopy-compatible proximity labeling

  • Split-BioID systems to detect transient ATG3 interactions during specific autophagy phases

• Live-cell imaging innovations:

  • FRET sensors to detect ATG3-substrate interactions in real-time

  • Optogenetic control of ATG3 activity to precisely manipulate autophagy spatially and temporally

  • Correlative light and electron microscopy (CLEM) to connect ATG3 fluorescence patterns with ultrastructural details

• Proteomics integration:

  • Antibody-based proximity proteomics to map the ATG3 interactome under different conditions

  • Tandem mass tag (TMT) labeling combined with ATG3 immunoprecipitation to quantify dynamic interaction changes

  • Parallel reaction monitoring (PRM) for targeted quantification of ATG3 post-translational modifications

• Single-cell analysis:

  • Imaging mass cytometry using metal-conjugated ATG3 antibodies for single-cell autophagy profiling in tissues

  • Single-cell Western blotting to analyze ATG3 expression heterogeneity

  • Flow cytometry with phospho-specific ATG3 antibodies to correlate ATG3 modification with autophagy stages

• Cryo-electron tomography:

  • Immunogold labeling with ATG3 antibodies for precise localization at autophagosome formation sites

  • Correlative cryo-fluorescence and cryo-electron microscopy to connect ATG3 dynamics with membrane remodeling

• Genome engineering approaches:

  • CRISPR-Cas9 knock-in of epitope tags for consistent antibody detection of endogenous ATG3

  • Base editing to introduce specific mutations in ATG3 (e.g., D156A, M157A) to study structure-function relationships

  • Endogenous tagging with split fluorescent proteins to visualize native ATG3 interactions

These innovative approaches, when combined with high-quality ATG3 antibodies, will enable researchers to address fundamental questions about ATG3's roles in autophagy with unprecedented precision and detail.