ATG16L1 Antibody

What is ATG16L1 and why is it important in autophagy research?

ATG16L1 (autophagy related 16 like 1) is a 607 amino acid protein with a molecular mass of approximately 68.3 kDa that plays a crucial role in both canonical and non-canonical autophagy pathways. It functions by interacting with the ATG12-ATG5 complex to mediate the lipidation of ATG8 family proteins (including MAP1LC3A, MAP1LC3B, MAP1LC3C, GABARAPL1, GABARAPL2, and GABARAP), which is essential for autophagosome formation . The protein's subcellular localization spans membranes, lysosomes, and cytoplasm, making it a key structural component in the autophagy machinery. Its importance in research is heightened by its association with inflammatory bowel disease, positioning it as both a marker for autophagy and a potential therapeutic target .

What are the major structural domains of ATG16L1 and how do they affect antibody selection?

Human ATG16L1 contains three major structural domains: the N-terminal ATG5 binding domain (ATG5-BD), the central coiled-coil domain (CCD), and the C-terminal WD40-domain . This domain organization is crucial when selecting antibodies, as epitopes located in different domains may yield varying results depending on the experimental context. For instance, antibodies targeting the N-terminal domain may be better suited for studying ATG16L1-ATG5 interactions, while those recognizing the WD40 domain might be preferable for examining interactions with downstream effectors. Researchers should select antibodies that target domains relevant to their specific research questions, considering that post-translational modifications like phosphorylation and protein cleavage can affect epitope accessibility in different domains .

How do ATG16L1 isoforms differ and how should this influence experimental design?

ATG16L1 exists in multiple isoforms, with ATG16L1α (63 kDa) and ATG16L1β (71 kDa) being the predominant variants expressed in intestinal epithelium and macrophages . All isoforms contain exon 9, which includes the important Thr300 residue. The presence of multiple isoforms necessitates careful experimental design, particularly when analyzing tissue-specific expression patterns. Researchers should select antibodies capable of detecting all relevant isoforms or use isoform-specific antibodies depending on their research objectives. When interpreting Western blot results, the observed molecular weight (ranging from 63-71 kDa) should be considered in the context of which isoforms might be present in the specific cell or tissue type under investigation .

What species reactivity should be considered when selecting an ATG16L1 antibody?

ATG16L1 is highly conserved across species, with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken . When selecting antibodies, it's essential to verify the species reactivity for your particular experimental model. For example, the polyclonal antibody (19812-1-AP) shows confirmed reactivity with human and mouse samples, while it has been cited for use with rat and pig samples as well . Cross-species reactivity can be advantageous for comparative studies but may also introduce complications in specificity. Researchers should review the validation data for their antibody of choice across multiple species, especially when working with less common model organisms or when translating findings between species .

What are the optimal conditions for using ATG16L1 antibodies in Western blotting?

For optimal Western blotting with ATG16L1 antibodies, several parameters require careful consideration. Based on the polyclonal antibody 19812-1-AP as an example, recommended dilutions range from 1:200 to 1:1000 . The expected molecular weight should be approximately 63-71 kDa, corresponding to the major isoforms ATG16L1α and ATG16L1β. Optimization should include evaluation of different blocking agents (typically 5% non-fat dry milk or BSA), incubation times (often overnight at 4°C for primary antibody), and detection methods. Positive controls such as MCF-7 cells, Jurkat cells, HEK-293T cells, or mouse spleen tissue can help validate experimental conditions, as these have been demonstrated to express detectable levels of ATG16L1 . When using phospho-specific ATG16L1 antibodies, special consideration should be given to phosphatase inhibitors during sample preparation to preserve the phosphorylation state .

How can phospho-ATG16L1 antibodies be utilized to monitor autophagy induction?

Phospho-ATG16L1 antibodies represent a powerful tool for monitoring autophagy induction, particularly because phospho-ATG16L1 is present only on newly forming autophagosomes . This characteristic makes these antibodies especially valuable for studying early events in autophagy, as their signal is not affected by prolonged stress or late-stage autophagy blocks that can confound other autophagy markers. To effectively utilize these antibodies, researchers should:

• Ensure proper sample preparation with phosphatase inhibitors

• Compare phospho-ATG16L1 levels across different time points after autophagy induction

• Correlate phospho-ATG16L1 signals with other autophagy markers for validation

• Consider that ATG16L1 phosphorylation represents a conserved signaling pathway activated by multiple autophagy-inducing stressors

These antibodies have been validated for Western blot, immunofluorescence, and immunohistochemistry applications, making them versatile tools for quantifying autophagy induction across different experimental platforms .

What controls should be included when validating ATG16L1 antibody specificity?

Validating ATG16L1 antibody specificity requires multiple complementary approaches. Essential controls include:

• Positive controls: Use cell lines with confirmed ATG16L1 expression such as MCF-7, Jurkat, or HEK-293T cells

• Negative controls: Include ATG16L1 knockout or knockdown samples when possible

• Peptide competition assays: Preincubate the antibody with immunizing peptide to confirm specific binding

• Cross-validation: Compare results across multiple antibodies targeting different epitopes of ATG16L1

• Species controls: Test reactivity across species if conducting comparative studies

For enhanced validation, researchers can employ orthogonal validation methods, comparing antibody-based detection with other protein detection techniques, or utilize independent antibody validation by comparing antibodies directed toward different epitopes of the same protein . These approaches ensure that signals detected truly represent ATG16L1 rather than non-specific binding.

What sample preparation methods are optimal for immunofluorescence with ATG16L1 antibodies?

For successful immunofluorescence with ATG16L1 antibodies, sample preparation is critical. Based on validated protocols:

• Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature preserves ATG16L1 structure while maintaining cellular architecture

• Permeabilization: 0.1-0.2% Triton X-100 for 5-10 minutes enables antibody access to cytoplasmic, membrane, and lysosomal pools of ATG16L1

• Blocking: 1-5% BSA or normal serum matching the secondary antibody species for 30-60 minutes

• Antibody incubation: For monoclonal antibodies like 1F12, dilutions should be optimized based on signal-to-noise ratio

• Counterstaining: Include markers for relevant subcellular compartments (e.g., lysosomal markers) to assess colocalization

When studying autophagosome formation, phospho-specific ATG16L1 antibodies can be particularly informative as they specifically label newly forming autophagosomes, allowing for precise temporal analysis of autophagy induction .

How can researchers differentiate between canonical and non-canonical autophagy using ATG16L1 antibodies?

ATG16L1 participates in both canonical and non-canonical autophagy pathways, making it a valuable marker for distinguishing between these processes. To differentiate between these pathways:

• Use co-immunoprecipitation with ATG16L1 antibodies to analyze binding partners specific to each pathway. In canonical autophagy, ATG16L1 strongly associates with ATG12-ATG5 complexes, whereas non-canonical pathways may show different interaction profiles .

• Implement dual immunostaining approaches combining ATG16L1 antibodies with markers specific to canonical (e.g., LC3B) or non-canonical (e.g., LAP-specific markers) pathways.

• Analyze the subcellular localization patterns of ATG16L1. In canonical autophagy, ATG16L1 localizes to pre-autophagosomal structures, while in non-canonical pathways, its distribution may differ.

• Utilize phospho-specific ATG16L1 antibodies, as phosphorylation states may differ between canonical and non-canonical pathways, providing a biochemical means of distinguishing these processes .

By systematically analyzing these parameters, researchers can determine the relative contribution of different autophagy pathways in their experimental system.

What approaches are effective for studying ATG16L1 involvement in inflammatory bowel disease?

Given the established association between ATG16L1 and inflammatory bowel disease (IBD) , several specialized approaches using ATG16L1 antibodies can advance this research:

• Tissue microarray analysis: Compare ATG16L1 expression patterns in normal versus IBD patient tissues using immunohistochemistry with validated ATG16L1 antibodies.

• Genetic variant studies: Combine antibody-based detection with genotyping to correlate protein expression/localization with disease-associated variants, particularly the T300A polymorphism.

• Cell type-specific analysis: Use flow cytometry or immunofluorescence with ATG16L1 antibodies in combination with cell type-specific markers to assess expression patterns in epithelial cells versus immune cells in intestinal tissues.

• Functional assays: Measure autophagy induction in response to bacterial stimuli in cells with different ATG16L1 genotypes using phospho-ATG16L1 antibodies .

These approaches can help elucidate how ATG16L1 variants contribute to IBD pathogenesis through altered autophagy regulation or other cellular processes.

How can researchers quantitatively assess ATG16L1-dependent autophagy in complex samples?

Quantitative assessment of ATG16L1-dependent autophagy requires robust methodological approaches:

• Multiplexed immunofluorescence: Combine ATG16L1 antibodies with other autophagy markers (LC3, p62) to quantify colocalization events as indicators of active autophagy.

• Phospho-ATG16L1 quantification: Measure phospho-ATG16L1 levels, which directly correspond to autophagy rates and are specifically associated with newly forming autophagosomes .

• Image-based high-content analysis: Implement automated quantification of ATG16L1-positive puncta number, size, and intensity in fluorescence microscopy images.

• Flow cytometry: For single-cell quantification in heterogeneous populations, combine surface markers with intracellular staining for ATG16L1.

• Tissue analysis algorithms: For immunohistochemistry in tissues, develop scoring systems that account for intensity, distribution, and cell type-specific expression of ATG16L1.

These quantitative approaches enable objective comparison of ATG16L1-dependent autophagy across experimental conditions or patient samples.

What are the considerations for studying post-translational modifications of ATG16L1?

ATG16L1 undergoes several post-translational modifications that affect its function, including phosphorylation and proteolytic cleavage . To effectively study these modifications:

• For phosphorylation studies:

  • Use phospho-specific antibodies like those detecting phospho-ATG16L1

  • Include phosphatase inhibitors during sample preparation

  • Consider λ-phosphatase treatment as a negative control

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms

• For proteolytic cleavage analysis:

  • Select antibodies recognizing epitopes that remain intact after cleavage

  • Use protease inhibitor cocktails during sample preparation

  • Compare molecular weight shifts under different conditions

  • Consider domain-specific antibodies to identify which regions are affected

• For temporal dynamics:

  • Implement time-course experiments after autophagy induction

  • Correlate modification patterns with functional outcomes

These approaches enable detailed characterization of how post-translational modifications regulate ATG16L1 function in different physiological and pathological contexts.

How should researchers interpret variations in ATG16L1 molecular weight observed in Western blots?

Variations in ATG16L1 molecular weight observed in Western blots can arise from multiple factors that require careful interpretation:

• Isoform expression: The two major isoforms, ATG16L1α (63 kDa) and ATG16L1β (71 kDa), show different tissue and cell-type specific expression patterns . Tissue-specific or developmental regulation may result in different banding patterns.

• Post-translational modifications: Phosphorylation can cause mobility shifts, particularly when multiple phosphorylation sites are occupied. Similarly, other modifications like ubiquitination can significantly alter apparent molecular weight.

• Proteolytic processing: ATG16L1 undergoes cleavage as part of its regulation, which can generate fragments of varying sizes depending on the cleavage site and cellular conditions.

• Sample preparation: Harsh lysis conditions or inadequate protease inhibitors may lead to artifactual degradation products.

To distinguish between these possibilities, researchers should:

• Compare results across different cell types with known isoform expression patterns

• Use phosphatase treatment to eliminate phosphorylation-dependent shifts

• Include appropriate molecular weight markers and positive controls

• Consider using multiple antibodies targeting different epitopes to verify results

What are common pitfalls in immunoprecipitation experiments with ATG16L1 antibodies?

When conducting immunoprecipitation (IP) with ATG16L1 antibodies, researchers should be aware of several common pitfalls:

• Insufficient antibody amount: For the polyclonal antibody 19812-1-AP, recommended usage is 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate . Using too little antibody can result in weak or undetectable precipitation.

• Cross-reactivity issues: ATG16L1 has multiple interaction partners in the autophagy pathway. Stringent washing conditions may be needed to reduce non-specific binding, but overly harsh conditions can disrupt legitimate protein-protein interactions.

• Buffer compatibility: The choice of lysis buffer can significantly impact IP efficiency. For membrane-associated proteins like ATG16L1, non-ionic detergents like NP-40 or Triton X-100 at 0.5-1% are often effective.

• Epitope masking: ATG16L1's interactions with ATG5-ATG12 or other proteins may mask epitopes, reducing antibody accessibility. Consider using antibodies targeting different epitopes if initial attempts fail.

• Denaturation sensitivity: Some antibodies perform poorly under denaturing conditions, making native IP preferable for certain applications.

To optimize IP protocols, researchers should validate their approach using positive controls like MCF-7 cells, which have been confirmed to yield positive IP results with ATG16L1 antibodies .

How can researchers troubleshoot weak or non-specific signals in immunohistochemistry with ATG16L1 antibodies?

Troubleshooting weak or non-specific signals in immunohistochemistry (IHC) with ATG16L1 antibodies requires systematic optimization:

• Antigen retrieval: ATG16L1 detection often benefits from heat-induced epitope retrieval. Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0) to determine optimal conditions.

• Antibody concentration: Titrate antibody dilutions to find the optimal balance between specific signal and background. For polyclonal antibodies, higher dilutions (1:500-1:1000) may reduce non-specific binding.

• Blocking optimization: Extend blocking times (up to 2 hours) or test alternative blocking agents (e.g., 5% normal serum, commercial blocking solutions) to reduce background.

• Detection system sensitivity: For weakly expressed targets, switch to more sensitive detection systems like polymer-based HRP systems or tyramide signal amplification.

• Tissue fixation: Over-fixation can mask epitopes while under-fixation can compromise tissue morphology. Standardize fixation protocols or test multiple fixation durations.

• Positive controls: Include tissues known to express ATG16L1, such as intestinal epithelium or lymphoid tissues, to validate staining protocols.

For phospho-specific ATG16L1 antibodies, additional considerations include using phosphatase inhibitors during tissue collection and processing, and potentially including phosphatase-treated sections as negative controls .

What approaches can verify that observed ATG16L1 signals correspond to authentic autophagy events?

To verify that observed ATG16L1 signals genuinely reflect autophagy events rather than non-specific antibody binding or autophagy-independent ATG16L1 functions:

• Correlation with multiple autophagy markers: Validate ATG16L1 signals by co-staining for additional autophagy proteins (LC3, p62, WIPI2) and assessing colocalization at the single-cell level.

• Autophagy modulation: Compare ATG16L1 patterns under basal conditions versus autophagy induction (starvation, rapamycin) and inhibition (Bafilomycin A1, Chloroquine).

• Genetic validation: Use ATG16L1 knockdown/knockout cells as negative controls and rescue experiments with wildtype ATG16L1 to confirm specificity.

• Phospho-ATG16L1 specificity: Utilize phospho-specific antibodies that selectively detect ATG16L1 on forming autophagosomes, as phospho-ATG16L1 signals directly correspond to autophagy rates and are not affected by late-stage autophagy blocks .

• Ultrastructural correlation: For definitive validation, combine immunogold labeling for ATG16L1 with electron microscopy to directly visualize association with autophagosomal structures.

These complementary approaches can provide robust confirmation that ATG16L1 signals correspond to legitimate autophagy processes rather than experimental artifacts or autophagy-independent functions.