ATG12A Antibody

What is ATG12 and why is it important in research?

ATG12 (Autophagy Related 12) is a critical protein in the autophagy pathway, functioning as a ubiquitin-like protein involved in autophagosome formation. It forms a conjugate with ATG5 through a ubiquitin-like conjugating system that includes ATG7 (E1-like activating enzyme) and ATG10 (E2-like conjugating enzyme) . The ATG12-ATG5 conjugate functions as an E3-like enzyme required for lipidation of ATG8 family proteins and their association with vesicle membranes . This protein is particularly significant in research because autophagy dysfunction is implicated in various diseases including cancer, neurodegenerative disorders, and infectious diseases . Current research indicates ATG12 may serve as a potential diagnostic and therapeutic target for hepatocellular carcinoma (HCC) .

What are the molecular characteristics of ATG12 that researchers should know?

When designing experiments with ATG12 antibodies, researchers should be aware of the following molecular characteristics:

• Calculated molecular weight: 15 kDa (free form)

• Observed molecular weight: 48-55 kDa (ATG12-ATG5 conjugate)

• Free ATG12 can appear as 15 kDa or 8 kDa bands in some experimental conditions

• Cellular localization: Cytoplasm and preautophagosomal structure membrane

• At least two isoforms of ATG12 are known to exist

• Gene ID (NCBI): 9140 (human)

• UniProt ID: O94817 (human)

What types of ATG12 antibodies are available for research applications?

Most commercially available ATG12 antibodies are polyclonal rabbit antibodies, which offer several advantages in research applications. These antibodies typically:

Polyclonal antibodies recognize multiple epitopes, making them useful for detecting proteins with post-translational modifications or different conformational states .

What are the optimal applications for ATG12 antibodies and their recommended dilutions?

ATG12 antibodies can be utilized across multiple experimental applications with specific dilution recommendations:

Researchers should validate these dilutions for their specific experimental systems, as optimal concentrations may vary depending on sample type, detection method, and antibody lot .

How should I design Western blot experiments to accurately detect ATG12?

When designing Western blot experiments to detect ATG12, consider the following methodological approach:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent protein degradation.

• Protein loading: Load 20-30 μg of whole cell lysate per lane for optimal detection .

• Gel selection: 12% SDS-PAGE gels are recommended for proper separation .

• Molecular weight markers: Include markers that cover both 15 kDa (free ATG12) and 48-55 kDa (ATG12-ATG5 conjugate) ranges .

• Transfer conditions: Standard PVDF or nitrocellulose membranes with wet transfer are suitable.

• Blocking: Use 5% non-fat milk or BSA in TBST.

• Primary antibody: Dilute ATG12 antibody 1:500-1:1000 in blocking buffer and incubate overnight at 4°C .

• Controls: Include positive control cell lines such as PC-3, COLO 320, NIH/3T3, HeLa, or HCT 116 cells .

• Interpretation: Expect bands at ~15 kDa (free ATG12) and/or 48-55 kDa (ATG12-ATG5 conjugate) .

What is the recommended protocol for immunohistochemistry with ATG12 antibodies?

For optimal immunohistochemical detection of ATG12 in tissue samples:

• Tissue preparation: Fix tissues in 10% neutral buffered formalin and embed in paraffin.

• Sectioning: Cut sections at 4-6 μm thickness.

• Deparaffinization: Standard xylene and graded ethanol series.

• Antigen retrieval: Use TE buffer (pH 9.0) or citrate buffer (pH 6.0) for 15 minutes. The choice depends on tissue type and preservation quality .

• Endogenous peroxidase blocking: 3% hydrogen peroxide for 10 minutes.

• Primary antibody: Dilute ATG12 antibody 1:50-1:500 and incubate according to your established protocol (typically overnight at 4°C or 1-2 hours at room temperature) .

• Detection system: Use appropriate secondary antibody and visualization system (DAB or other chromogens).

• Counterstaining: Hematoxylin is typically used.

• Controls: Include both positive control tissues (human colon cancer tissue has been validated ) and negative controls (primary antibody omission).

How do I interpret the different molecular weight bands when detecting ATG12?

Researchers often encounter multiple bands when detecting ATG12 by Western blot, which require careful interpretation:

• 15 kDa band: Represents free, unconjugated ATG12 protein

• 8 kDa band: May represent an alternative form of free ATG12 in some experimental conditions

• 48-55 kDa band: Represents the ATG12-ATG5 conjugate, which is the functionally active form involved in autophagy

The relative abundance of these bands can provide insights into autophagy status:

• An increase in the ATG12-ATG5 conjugate (48-55 kDa) often indicates autophagy activation

• Changes in the ratio between free and conjugated forms may reflect alterations in autophagy dynamics

• Complete absence of the conjugate form may indicate defects in the ATG12-ATG5 conjugation machinery

How can I validate the specificity of ATG12 antibody signals in my experiments?

To ensure the specificity of ATG12 antibody signals and avoid misinterpretation:

• Knockdown/knockout validation: Use ATG12 knockdown or knockout samples as negative controls. Published knockdown/knockout validation data is available for some antibodies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide, which should abolish specific signals.

• Multiple antibody approach: Use antibodies from different sources or targeting different epitopes of ATG12.

• Molecular weight verification: Confirm that observed bands align with expected molecular weights (15 kDa for free ATG12, 48-55 kDa for ATG12-ATG5 conjugate) .

• Positive controls: Include cell lines known to express ATG12, such as PC-3, COLO 320, NIH/3T3, HeLa, or HCT 116 cells .

• Recombinant protein: Compare signals with purified recombinant ATG12 protein when available.

What controls should I include when studying ATG12 in autophagy research?

Comprehensive control strategies for ATG12 in autophagy research include:

• Autophagy induction controls:

  • Starvation (EBSS or serum-free media) as a positive control for autophagy induction

  • Rapamycin treatment (mTOR inhibitor) to induce autophagy

• Autophagy inhibition controls:

  • Bafilomycin A1 or chloroquine to block autophagosome-lysosome fusion

  • 3-Methyladenine (3-MA) to inhibit early autophagy

• ATG protein controls:

  • Monitor other autophagy markers (LC3-I/II, p62/SQSTM1) in parallel with ATG12

  • Examine ATG5 levels, as ATG12 functions primarily in the ATG12-ATG5 conjugate

• Cell/tissue type considerations:

  • Include both autophagy-competent and autophagy-deficient cells (e.g., ATG5-/- or ATG7-/-)

  • Compare normal vs. disease tissues when relevant

• Experimental timing:

  • Establish a time course to capture dynamic changes in ATG12 expression and conjugation

How can I study the ATG12-ATG5 conjugation mechanism in my experimental system?

To investigate the ATG12-ATG5 conjugation process:

• Co-immunoprecipitation approach:

  • Use ATG12 antibodies to immunoprecipitate the protein complex

  • Perform Western blot analysis with ATG5 antibodies to detect the conjugate

  • Include ATG7 and ATG10 detection to analyze the entire conjugation machinery

• Genetic manipulation strategy:

  • Overexpress tagged versions of ATG12 and ATG5 (e.g., FLAG-ATG12 and HA-ATG5)

  • Introduce mutations at the conjugation site (G140 in human ATG12) to disrupt conjugation

  • Use siRNA or CRISPR/Cas9 to knockdown/knockout ATG7 or ATG10 to inhibit conjugation

• In vitro reconstitution:

  • Establish an in vitro system with purified components (ATG12, ATG5, ATG7, ATG10)

  • Monitor conjugate formation under various conditions (ATP, pH, temperature)

  • Introduce potential inhibitors or enhancers of the conjugation process

• Structural analysis:

  • Use protein structural prediction tools to identify critical interaction domains

  • Design domain-specific antibodies or blocking peptides to interfere with specific steps

What are common troubleshooting strategies for ATG12 detection issues?

When facing detection problems with ATG12 antibodies:

How can I investigate ATG12's role in non-canonical functions beyond autophagy?

ATG12 has several functions beyond its canonical role in autophagy. To investigate these:

• Innate immune response regulation:

  • Study ATG12's interaction with RIG-I and VISA/IPS-1 through co-immunoprecipitation

  • Measure type I interferon production during viral infection in ATG12-manipulated cells

  • Analyze viral replication efficacy in cells with modified ATG12 expression

• Hepatitis C virus (HCV) replication:

  • Use ATG12 knockdown/overexpression in HCV infection models

  • Monitor HCV RNA translation in the presence/absence of ATG12

  • Differentiate between ATG12's roles in initial infection versus established infection

• Cell death pathways:

  • Examine interactions between ATG12 and apoptotic proteins

  • Analyze cell death patterns in ATG12-deficient versus ATG12-overexpressing cells

  • Use specific inhibitors of different cell death pathways to determine ATG12 involvement

• Cancer progression mechanisms:

  • Investigate ATG12 expression patterns in tumor versus normal tissues

  • Correlate ATG12 levels with clinical outcomes in cancer patients

  • Develop experimental models to test ATG12 as a therapeutic target in HCC and other cancers

What is the potential of ATG12 as a therapeutic target in cancer research?

Current research indicates ATG12 may serve as an important therapeutic target:

• Hepatocellular carcinoma (HCC) implications:

  • Recent studies suggest ATG12 plays a significant role in HCC progression

  • ATG12 may serve as both a diagnostic marker and therapeutic target for HCC

  • Research is exploring the molecular mechanisms of ATG12's contribution to hepatocarcinogenesis

• Therapeutic approaches under investigation:

  • Small molecule inhibitors of the ATG12-ATG5 conjugation pathway

  • Targeted degradation strategies (PROTACs) directed at ATG12

  • Combination therapies targeting both ATG12 and complementary oncogenic pathways

• Diagnostic potential:

  • ATG12 expression patterns may serve as prognostic indicators

  • The ATG12-ATG5 conjugate levels could potentially distinguish aggressive from indolent tumors

  • Liquid biopsy approaches may detect circulating ATG12 or its fragments

• Research challenges:

  • Distinguishing pro-survival versus pro-death roles of autophagy in different cancer stages

  • Developing selective targeting approaches that don't disrupt essential autophagy

  • Understanding compensatory mechanisms that might emerge after ATG12 inhibition

How can I design experiments to study ATG12's role in selective autophagy processes?

To investigate ATG12's involvement in selective autophagy:

• Cargo-specific autophagy experimental design:

  • Mitophagy: Compare PINK1/Parkin-mediated mitochondrial clearance in ATG12-manipulated cells

  • Xenophagy: Assess intracellular bacterial clearance rates and colocalization with autophagy markers

  • Aggrephagy: Monitor clearance of protein aggregates in neurodegenerative disease models

• Receptor interaction studies:

  • Analyze ATG12's potential interactions with selective autophagy receptors (p62, NBR1, OPTN)

  • Perform proximity ligation assays to detect in situ interactions

  • Develop fluorescently tagged constructs to visualize dynamics in live cells

• Structural determination approaches:

  • Use cryo-EM or X-ray crystallography to resolve ATG12-ATG5 interactions with cargo receptors

  • Perform in silico modeling to predict binding sites and interaction domains

  • Design mutants with altered binding capacities for functional validation

• Systems biology integration:

  • Conduct proteomics to identify the complete interactome of ATG12 under different conditions

  • Employ CRISPR screens to identify genetic modifiers of ATG12's selective autophagy functions

  • Develop computational models that predict ATG12's role in various selective autophagy pathways

What are the latest methodological advances for studying ATG12 in autophagy research?

Emerging technologies are expanding our ability to study ATG12:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize ATG12 localization with nanometer precision

  • Live-cell imaging with split fluorescent protein systems to monitor ATG12-ATG5 conjugation in real-time

  • Correlative light and electron microscopy (CLEM) to study ATG12's role in autophagosome formation

• Proteomics approaches:

  • Proximity-dependent biotin identification (BioID) to map the ATG12 interaction network

  • Quantitative mass spectrometry to measure ATG12 modifications and turnover rates

  • Crosslinking mass spectrometry to identify structural interfaces in the ATG12-ATG5 complex

• Genetic engineering advancements:

  • CRISPR base editing for introducing specific ATG12 mutations without double-strand breaks

  • Inducible degradation systems for temporal control of ATG12 protein levels

  • Cell-type specific conditional knockouts for in vivo studies of ATG12 function

• Single-cell technologies:

  • Single-cell transcriptomics to analyze ATG12 expression heterogeneity in tissues

  • Single-cell proteomics to measure ATG12 protein levels and modifications at cellular resolution

  • Microfluidic approaches to analyze autophagy dynamics in individual cells with varying ATG12 levels