ATG18H Antibody

What is ATG18 and why is it important in autophagy research?

ATG18 is an essential protein involved in both autophagy and the regulation of vacuolar morphology. It forms a complex with ATG2 and binds to phosphatidylinositol 3-phosphate (PtdIns(3)P), which is crucial for its localization to autophagic membranes. ATG18 is a key component in both selective and non-selective autophagy pathways, making it an important target for autophagy research . The protein contains an FRRG motif (residues 284-287) that is critical for phosphoinositide binding. When this binding is disrupted, as in the ATG18(FTTG) variant, proper localization to autophagic membranes is compromised, resulting in reduced autophagic activity . Understanding ATG18's role provides valuable insights into the molecular mechanisms of autophagy and its regulation.

How do researchers distinguish between ATG18H antibody specificity and cross-reactivity with related proteins?

Researchers can employ several methodological approaches to assess ATG18H antibody specificity. First, validation in knockout or knockdown systems is essential - comparing antibody reactivity in wild-type versus ATG18-deficient samples provides critical information about specificity . For cross-reactivity concerns, especially with related PROPPIN family proteins, western blotting against recombinant proteins can reveal potential cross-reactivity patterns. Additionally, immunoprecipitation followed by mass spectrometry can identify all proteins captured by the antibody .

The gold standard approach combines multiple validation methods:

• Using multiple antibodies targeting different epitopes of ATG18

• Employing peptide competition assays to confirm epitope specificity

• Testing against a panel of related proteins expressed in vitro

• Performing immunostaining with co-localization studies using established markers

• Comparing reactivity in different species if the antibody is designed for cross-species detection

What are the preferred fixation and sample preparation methods when using ATG18H antibodies for immunohistochemistry (IHC)?

For optimal IHC results with ATG18H antibodies, sample preparation is critical. Paraformaldehyde fixation (4%) for 10-15 minutes typically preserves ATG18 epitopes while maintaining cellular architecture . Notably, rabbit-derived ATG18H antibodies often demonstrate superior sensitivity in IHC applications compared to mouse monoclonal antibodies targeting the same antigen, as multiple studies have consistently demonstrated higher sensitivity of rabbit mAbs in IHC applications .

For membrane permeabilization, a gentle approach using 0.1-0.3% Triton X-100 for 5-10 minutes is recommended to maintain the integrity of autophagic membranes while allowing antibody access . When visualizing ATG18 localization to autophagy-related structures, it's advisable to:

• Include starvation or rapamycin treatment conditions to enhance autophagy induction

• Use antigen retrieval methods (citrate buffer, pH 6.0) for formalin-fixed samples

• Block with 3-5% BSA or serum matching the secondary antibody species

• Incubate with primary antibody overnight at 4°C

• Employ fluorescent secondary antibodies for co-localization studies

How can I monitor ATG18-ATG2 complex formation using ATG18H antibodies?

Co-immunoprecipitation (co-IP) assays using ATG18H antibodies provide a reliable method to monitor ATG18-ATG2 complex formation. Based on established protocols, researchers should:

• Treat cells with an autophagy inducer (e.g., rapamycin for 10 minutes) to enhance complex formation

• Generate spheroplasts while maintaining cells in rapamycin-containing buffers

• Lyse spheroplasts by osmotic shock in immunoprecipitation buffer (50 mM Tris-HCl pH 8.0, 200 mM sorbitol, 150 mM KCl, 5 mM MgCl₂, 0.5 mg/ml BSA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors

• Remove cellular debris by centrifugation at 500 × g for 5 minutes

• Centrifuge samples at 100,000 × g for 30 minutes

• Incubate supernatants with anti-ATG2 or anti-ATG18H antibodies

• Analyze by western blotting using both anti-ATG18 and anti-ATG2 antibodies

Importantly, research has demonstrated that ATG18-ATG2 complex formation occurs independently of PtdIns(3)P binding, as the ATG18(FTTG) variant incapable of binding phosphoinositides still forms a complex with ATG2 at similar efficiency to wild-type ATG18 .

How does the binding affinity of ATG18H antibodies compare between rabbit and mouse-derived versions, and what implications does this have for research applications?

Rabbit-derived ATG18H antibodies typically demonstrate superior binding characteristics compared to their mouse counterparts. Extensive comparative studies of rabbit and mouse monoclonal antibodies have revealed that rabbit mAbs consistently show higher sensitivity in immunohistochemistry applications . This difference stems from the unique ontogeny of rabbit B cells, which produces antibody repertoires with distinctive characteristics.

In quantitative affinity studies, rabbit monoclonal antibodies targeting various antigens display affinity ranges of 20-200 pM (median 66 pM), with some exceptional antibodies reaching near the detection limit of 1 pM . While mouse mAbs show similar median affinity values (72 pM), rabbit antibodies often demonstrate superior performance in complex applications due to:

• Higher diversity in complementarity-determining regions (CDRs)

• More efficient in vivo maturation and selection processes

• Superior ability to recognize epitopes that might be immunologically silent in mice

For ATG18H research applications, this translates to:

• Enhanced detection sensitivity in low-expression systems

• Better recognition of native conformations

• Superior performance in fixed tissue samples

• Improved epitope accessibility in complex protein structures

Researchers should consider these advantages when selecting antibodies for challenging applications like detecting transient ATG18 recruitment to forming autophagosomes .

What methodological approaches can resolve contradictory localization data when using ATG18H antibodies in autophagy research?

When faced with contradictory ATG18 localization data, a structured methodological approach can resolve discrepancies:

• Antibody validation confirmation: Verify antibody specificity using knockout/knockdown controls and peptide competition assays.

• Cell-specific expression profiling: Quantify ATG18 expression levels across cell types using RT-qPCR and western blotting to identify potential isoform or expression level differences.

• Stimulus-dependent localization analysis: Systematically analyze ATG18 localization under different autophagy induction methods:

  • Nutrient starvation (S (-NC) medium lacking both nitrogen and carbon sources)

  • Rapamycin treatment

  • Oxidative stress

  • ER stress inducers

• Multi-method localization confirmation:

  • Fluorescent protein tagging (C-terminal HA-GFP fusion)

  • Proximity labeling approaches (BioID or APEX2)

  • Super-resolution microscopy

  • Subcellular fractionation followed by western blotting

• Dynamic recruitment studies: Implement live-cell imaging with ATG18H-GFP fusions to track temporal localization patterns during autophagosome formation.

Research has established that ATG18 localizes to multiple membrane structures, including endosomes, the vacuolar membrane, and autophagic membranes, while the PtdIns(3)P-binding deficient ATG18(FTTG) variant fails to localize to these structures . This indicates that PtdIns(3)P binding is crucial for proper ATG18 localization during autophagy.

How can I optimize ATG18H antibodies for detecting the phosphoinositide-binding deficient forms of ATG18?

Detecting phosphoinositide-binding deficient forms of ATG18, such as the ATG18(FTTG) variant, requires specific optimization strategies:

• Epitope-specific antibody selection: Choose antibodies targeting regions distant from the FRRG motif (residues 284-287) to ensure the mutation doesn't interfere with antibody recognition.

• Increased antibody concentration: The ATG18(FTTG) variant often requires 1.5-2× higher antibody concentrations due to its altered cellular distribution.

• Modified extraction buffers: Include stronger detergents (0.5% Triton X-100 with 0.1% SDS) to enhance extraction efficiency of mislocalized ATG18(FTTG).

• Alternative detection strategies: Consider:

  • HA-tagging ATG18(FTTG) and using anti-HA antibodies

  • C-terminal GFP fusion proteins for direct fluorescence detection

  • Employing the 2×FYVE domain fusion approach, which restores proper localization

• Control experiments: Include side-by-side comparisons with wild-type ATG18 and utilize the ATG18(FTTG)-HA-2×FYVE construct, which artificially restores PtdIns(3)P binding through the FYVE domain .

Research has demonstrated that the ATG18(FTTG)-HA-2×FYVE construct, where a specific PtdIns(3)P-binding domain (2×FYVE) is fused to ATG18(FTTG), restores proper localization to autophagic membranes and full autophagic activity . This suggests that the primary function of PtdIns(3)P binding is to localize the ATG18-ATG2 complex to appropriate membranes rather than altering the intrinsic activity of ATG18.

What complementary assays should be used alongside ATG18H antibody detection to comprehensively evaluate autophagy dynamics?

A comprehensive autophagy evaluation requires multiple complementary approaches alongside ATG18H antibody detection:

For ATG18-specific evaluation, researchers should:

• Monitor PtdIns(3)P levels using 2×FYVE domain probes to correlate with ATG18 recruitment

• Assess ATG2 localization, as ATG18 is required for proper ATG2 recruitment to autophagic membranes

• Employ autophagic body accumulation assays in S (-NC) medium lacking both nitrogen and carbon sources to visualize autophagy completion

• Measure the alkaline phosphatase (ALP) activity to quantitatively assess autophagic flux

• Analyze the maturation of precursor aminopeptidase I (prApeI) to evaluate the cytoplasm-to-vacuole targeting (Cvt) pathway

This multi-parameter approach ensures robust assessment of ATG18's various functions in autophagy regulation.

How can I overcome background issues when using ATG18H antibodies in immunofluorescence microscopy?

Background signal is a common challenge when using ATG18H antibodies for immunofluorescence. Implement these methodological strategies to optimize signal-to-noise ratio:

• Blocking optimization: Extend blocking time to 2 hours using a combination of 5% normal serum (matching secondary antibody species) with 3% BSA. Adding 0.1% Tween-20 to blocking solutions often reduces non-specific binding.

• Antibody dilution optimization: Perform a dilution series (1:100 to 1:2000) with both primary and secondary antibodies to identify optimal concentrations. For rabbit monoclonal ATG18H antibodies, starting at 1:500 dilution is typically effective .

• Sample preparation refinement:

  • Reduce autofluorescence with 0.1% sodium borohydride treatment (10 minutes) before blocking

  • For tissue sections, include a 0.3% H₂O₂ treatment to neutralize endogenous peroxidases

  • Consider using Sudan Black B (0.1% in 70% ethanol) to quench lipofuscin autofluorescence

• Washing protocol enhancement: Implement extended washing steps (5 × 5 minutes) with PBS containing 0.1% Tween-20 after both primary and secondary antibody incubations.

• Controls optimization:

  • Include ATG18-deficient samples as negative controls

  • Use competing peptides to confirm specificity

  • Include secondary-only controls to assess non-specific binding

For challenging samples, consider additional signal amplification methods like tyramide signal amplification or proximity ligation assays, which can enhance specific signals while maintaining low background.

What are the critical considerations when using ATG18H antibodies in human clinical samples versus model organisms?

When transitioning ATG18H antibody applications from model organisms to human clinical samples, several critical factors require adjustment:

• Species-specific epitope variations: Human WIPI2 (the mammalian homolog of yeast ATG18) exhibits specific epitope differences that necessitate antibodies raised against the human sequence. Antibodies recognizing yeast ATG18 often show minimal cross-reactivity with human WIPI proteins .

• Isoform specificity: Humans express multiple WIPI protein family members (WIPI1-4) and splice variants that must be distinguished. Verify antibody specificity against each isoform using recombinant protein panels.

• Fixation protocol optimization:

  • Clinical formalin-fixed paraffin-embedded (FFPE) tissues require more rigorous antigen retrieval (high-pressure or pH-optimized buffers)

  • Fresh frozen human tissues require shorter fixation times (10 minutes in 4% PFA)

  • Consider testing multiple fixation methods on control tissues

• Validation requirements:

  • For diagnostic applications, FDA-approved rabbit mAbs are available as precedents

  • Clinical applications require extensive specificity testing across tissue panels

  • Include appropriate disease controls alongside normal tissues

• Technical considerations:

  • Human samples often demonstrate higher background autofluorescence requiring additional quenching steps

  • Patient variability necessitates larger sample sizes for reliable results

  • Tissue processing variations between clinical centers may affect antibody performance

The superior sensitivity of rabbit monoclonal antibodies makes them particularly valuable for clinical applications, as evidenced by the FDA approval of multiple rabbit mAbs for the detection of tumor-associated antigens .

How can I quantitatively assess the contribution of ATG18 to selective versus non-selective autophagy using ATG18H antibodies?

Quantitatively distinguishing ATG18's roles in selective versus non-selective autophagy requires a methodical approach using ATG18H antibodies alongside complementary assays:

• Pathway-specific substrate tracking:

  • For selective autophagy: Monitor prApeI processing via the Cvt pathway using anti-ApeI antibodies

  • For non-selective autophagy: Quantify bulk autophagic flux using the ALP assay under nitrogen starvation conditions

• Comparative analysis using ATG18 variants:

  • Wild-type ATG18 serves as reference

  • ATG18(FTTG) shows significantly reduced function in both pathways

  • ATG18(FTTG)-HA-2×FYVE restores function by artificial targeting

• Quantitative immunofluorescence:

  • Co-localization analysis of ATG18H with selective autophagy receptors (p62, NBR1)

  • Measure Pearson's correlation coefficients between ATG18 and autophagy markers

  • Quantify puncta formation under different induction conditions

• Biochemical fractionation:

  • Isolate autophagic membranes from cells under selective versus non-selective conditions

  • Quantify ATG18 recruitment by western blotting

  • Assess co-recruitment of pathway-specific factors

• Genetic interaction studies:

  • Combine ATG18 mutations with selective autophagy receptor mutations

  • Quantify epistatic relationships using autophagy flux assays

  • Measure rescue efficiencies using the ATG18(FTTG)-HA-2×FYVE construct

Research has demonstrated that PtdIns(3)P binding of ATG18 is essential for full activity in both selective and non-selective autophagy, with the ATG18(FTTG) variant showing significantly reduced function in both pathways . The restoration of function by targeting ATG18(FTTG) to PtdIns(3)P-enriched sites via the 2×FYVE domain indicates that proper membrane localization is the critical factor for ATG18 function in both autophagy modes .

How can ATG18H antibodies be utilized in the development of autophagy-modulating therapeutics?

ATG18H antibodies serve as valuable tools in developing autophagy-modulating therapeutics through several strategic applications:

• Target validation and mechanism studies:

  • Confirm ATG18/WIPI protein involvement in disease-relevant autophagy pathways

  • Characterize ATG18 expression patterns in pathological versus normal tissues

  • Identify specific points in the autophagy cascade amenable to therapeutic intervention

• High-throughput screening platforms:

  • Develop immunofluorescence-based assays to monitor ATG18 recruitment to autophagic structures

  • Create cell-based assays using ATG18H antibodies to identify compounds that modulate ATG18 function

  • Establish ELISA-based methods to quantify ATG18-ATG2 complex formation

• Therapeutic antibody development:

  • Though no therapeutic rabbit mAbs have yet received FDA approval, several are in clinical trials for oncology applications

  • Apply humanization strategies (similar to sevacizumab, APX005M, and YYB101) to ATG18-targeting antibodies

  • Develop ATG18-targeting antibodies conjugated to drugs or nanoparticles for targeted delivery

• Companion diagnostic development:

  • Utilize ATG18H antibodies as biomarkers to identify patients likely to respond to autophagy modulators

  • Develop IHC-based diagnostic tests similar to FDA-approved rabbit mAbs currently used for detecting tumor-associated antigens

  • Create multiplex assays combining ATG18H with other autophagy markers

The high sensitivity and specificity of rabbit-derived ATG18H antibodies make them particularly valuable for these applications, as demonstrated by the success of rabbit mAbs in diagnostic applications and their progression into clinical trials for therapeutic use .

What novel approaches can enhance the sensitivity of ATG18H antibodies for detecting transient autophagy-related structures?

Enhancing ATG18H antibody sensitivity for transient autophagy structures requires innovative techniques:

• Signal amplification strategies:

  • Quantum dot-conjugated secondary antibodies provide superior signal-to-noise ratios

  • Proximity ligation assays detect ATG18-ATG2 interactions with single-molecule sensitivity

  • Tyramide signal amplification enhances fluorescent signals up to 100-fold

• Advanced microscopy integration:

  • Super-resolution techniques (STORM, PALM, STED) resolve ATG18-positive structures below diffraction limit

  • Lattice light-sheet microscopy captures rapid dynamics of ATG18 recruitment

  • Correlative light-electron microscopy (CLEM) links fluorescence signals to ultrastructural context

• Genetic engineering approaches:

  • Split fluorescent protein complementation assays visualize ATG18-ATG2 interactions

  • HaloTag or SNAP-tag ATG18 fusions enable pulse-chase labeling of transient structures

  • Photoactivatable fluorescent protein fusions for optogenetic control and tracking

• Biochemical enhancements:

  • Crosslinking strategies to stabilize transient ATG18 associations

  • Optimized extraction buffers preserving labile membrane associations

  • Detergent-free isolation of membrane microdomains containing ATG18

• Computational analysis methods:

  • Machine learning algorithms to identify subtle ATG18-positive structures

  • Deconvolution and image processing to enhance signal detection

  • 4D tracking algorithms for temporal analysis of ATG18 dynamics

These approaches build upon the demonstrated importance of PtdIns(3)P binding for ATG18 localization to autophagic membranes and leverage the high sensitivity of rabbit-derived antibodies, which have consistently shown superior performance in detecting low-abundance targets .

What are the emerging trends in ATG18H antibody development and application for understanding autophagy regulation?

The field of ATG18H antibody development is advancing rapidly, with several emerging trends that will shape future autophagy research:

• Multiplexed antibody approaches: Simultaneous detection of multiple autophagy proteins (ATG18, ATG2, ATG9, etc.) using spectrally distinct fluorophores or mass cytometry is enabling more comprehensive analysis of autophagy dynamics and protein interactions.

• Site-specific phosphorylation detection: Development of phospho-specific ATG18H antibodies to detect regulatory modifications that may modulate PtdIns(3)P binding or ATG2 interaction is revealing new layers of autophagy regulation.

• Tissue and cell-type specific variant recognition: Increasingly specific antibodies targeting ATG18 isoforms or splice variants are uncovering cell-type specific autophagy regulation mechanisms in complex tissues.

• Conformation-specific antibodies: Novel antibodies that specifically recognize active versus inactive ATG18 conformations are providing insights into the structural changes that occur during membrane binding.

• Integration with systems biology approaches: Combining ATG18H antibody-based detection with proteomics, metabolomics, and transcriptomics is creating multi-dimensional datasets that reveal broader autophagy regulatory networks.

The exceptional specificity and sensitivity of rabbit-derived monoclonal antibodies position them at the forefront of these developments, with their continued advancement likely to fuel discoveries in basic autophagy mechanisms and therapeutic applications . As therapeutic rabbit mAb-derived agents advance through clinical trials, the technologies and knowledge gained will further enhance the development of ATG18H-related research and diagnostic tools .

How might understanding of ATG18's role in autophagy contribute to personalized medicine approaches to diseases with autophagy dysregulation?

ATG18's central role in autophagy positions it as a valuable target for personalized medicine approaches to diseases characterized by autophagy dysregulation:

• Biomarker development: ATG18H antibodies enable the identification of patient-specific autophagy signatures in:

  • Neurodegenerative diseases (Alzheimer's, Parkinson's)

  • Cancer progression and therapeutic resistance

  • Inflammatory and autoimmune conditions

  • Metabolic disorders

• Therapeutic targeting strategies:

  • Modulation of ATG18-PtdIns(3)P interaction as a specific control point

  • Targeting ATG18-ATG2 complex formation for pathway-specific regulation

  • Developing compounds that affect ATG18 recruitment to specific membrane compartments

• Patient stratification approaches:

  • ATG18 expression patterns may predict response to autophagy-modulating therapies

  • Genetic variants affecting ATG18 function could guide personalized treatment selection

  • Tissue-specific ATG18 recruitment profiles may indicate disease subtypes

• Monitoring therapeutic responses:

  • Serial sampling of patient tissues using ATG18H antibodies to track treatment efficacy

  • Development of circulating ATG18 detection methods as liquid biopsy approaches

  • Correlation of ATG18 function with disease progression markers