ATG16 Antibody

What is ATG16L1 and what is its significance in autophagy research?

ATG16L1 is a crucial autophagy-related protein that forms a complex with the ATG5-ATG12 conjugate. This multimeric protein is essential for autophagosome formation in both yeast and mammals, targeting the ATG5-ATG12 complex to the autophagic isolation membrane during autophagosome development . Human ATG16L1 is a 607 amino acid protein (~68 kDa) comprising three major domains: the N‐terminal ATG5 binding domain (ATG5‐BD), the central coiled‐coil domain (CCD), and a predicted C‐terminal WD40‐domain . It plays a vital role in preserving cellular nutrients under starvation conditions and facilitating the normal turnover of cytosolic components . The protein's central role in autophagy makes it an important target for researchers studying this fundamental cellular process.

What are the main structural features and isoforms of ATG16L1?

ATG16L1 contains several distinct domains that contribute to its function in autophagy:

• N-terminal ATG5 binding domain that facilitates interaction with the ATG5-ATG12 complex

• Central coiled-coil domain important for protein-protein interactions

• C-terminal WD40-domain with seven WD-repeats that forms a platform for further protein interactions

• An amphipathic α-helix (amino acid residues 113–131) with coiled-coil-like propensity that mediates membrane binding

The protein exists in multiple isoforms, with ATG16L1α (63 kDa) and ATG16L1β (71 kDa) being the major variants expressed in intestinal epithelium and macrophages. All isoforms encode exon 9, which contains the important Threonine 300 residue . The observed molecular weight ranges from 63-71 kDa depending on the specific isoform being detected .

Why are phospho-specific ATG16L1 antibodies particularly valuable for autophagy research?

Phospho-specific ATG16L1 antibodies represent an exciting advancement in autophagy research because they can detect endogenous phosphorylated ATG16L1, which is only present on newly forming autophagosomes . This characteristic provides significant advantages over traditional autophagy markers since phospho-ATG16L1 levels are not affected by prolonged stress or late-stage autophagy blocks, which can confound conventional autophagy analysis . Most importantly, measured phospho-ATG16L1 levels directly correspond to autophagy rates, making these antibodies particularly useful for monitoring autophagy induction in rare cell populations or in vivo settings . The phospho-antibody can be utilized across multiple applications including western blot, immunofluorescence, and immunohistochemistry, providing versatility for different experimental designs .

Experimental Applications and Methodological Approaches

For optimal ATG16L1 detection, sample preparation should follow these methodological guidelines:

For Western Blotting:

• Lyse cells or tissues in a buffer containing protease inhibitors to prevent degradation

• Use appropriate lysis conditions (e.g., RIPA buffer for total protein extraction)

• Load adequate protein amounts (typically 20-50 μg total protein)

• Separate proteins on 8-12% SDS-PAGE gels to properly resolve the 63-71 kDa bands

• Transfer to nitrocellulose or PVDF membranes using standard protocols

• Block with 5% non-fat milk or BSA in TBST

• Apply antibody at recommended dilutions (1:200-1:1000)

• Detect using appropriate secondary antibodies and visualization systems

For Immunofluorescence/Immunohistochemistry:

• Fix samples with 4% paraformaldehyde or other appropriate fixatives

• For paraffin sections, perform antigen retrieval as needed

• Permeabilize with 0.1-0.5% Triton X-100 for intracellular protein access

• Block nonspecific binding with appropriate blocking buffer

• Apply primary antibody at recommended dilutions and incubate overnight at 4°C

• Use species-appropriate secondary antibodies for detection

• Counter-stain nuclei with DAPI if desired

How should ATG16L1 antibodies be stored and handled to maintain optimal activity?

To ensure maximum efficacy and shelf life, ATG16L1 antibodies should be handled according to these guidelines:

• Store at -20°C in the provided storage buffer (typically PBS with 0.02% sodium azide and 50% glycerol at pH 7.3)

• Antibodies remain stable for one year after shipment when properly stored

• Minimize freeze-thaw cycles to prevent protein denaturation and loss of activity

• For some formulations, aliquoting is unnecessary for -20°C storage (verify with supplier information)

• When working with the antibody, keep on ice and return to storage promptly

• Some preparations may contain 0.1% BSA as a stabilizer

• For diluted working solutions, prepare fresh or store short-term at 4°C with preservatives

How do you select the appropriate ATG16L1 antibody for specific experimental questions?

Selection criteria should be based on the following considerations:

Epitope Specificity:
Different antibodies target distinct regions of ATG16L1, each with potential advantages:

• Antibodies targeting AA 161-190 region for general ATG16L1 detection

• Phospho-specific antibodies for autophagy induction studies

• Antibodies targeting AA 84-114, 11-257, or 501-607 for other domain-specific analyses

Species Reactivity:

• Some antibodies react only with human samples

• Others show cross-reactivity with human and mouse

• Some have broader reactivity including human, mouse, and rat

Antibody Format and Clonality:

• Polyclonal antibodies often provide higher sensitivity but lower specificity

• Monoclonal antibodies offer consistent specificity between lots

• Choose based on the level of specificity required and application needs

Validation Status:

• Review published applications data

• Check manufacturer validation for your specific application

• Consider antibodies with multiple validated applications for flexibility

What are common challenges in ATG16L1 antibody experiments and how can they be resolved?

Multiple Bands in Western Blot:

• Expected observation: ATG16L1 shows multiple isoforms between 63-71 kDa

• Potential issues: Non-specific binding, degradation products

• Solutions: Optimize blocking conditions, use fresh samples with protease inhibitors, adjust antibody concentration

Weak Signal:

• Potential causes: Low protein expression, insufficient antibody concentration, inefficient transfer

• Solutions: Increase protein loading, decrease antibody dilution (try 1:200 instead of 1:1000), optimize transfer conditions, consider more sensitive detection systems

High Background:

• Potential causes: Insufficient blocking, antibody concentration too high, inadequate washing

• Solutions: Increase blocking time/concentration, dilute antibody further, extend washing steps, use more specific secondary antibodies

Inconsistent Staining Patterns:

• Potential causes: Fixation artifacts, sample variability, antibody batch differences

• Solutions: Standardize fixation and preparation protocols, include positive controls (e.g., MCF-7 cells or mouse spleen tissue) , compare results with multiple ATG16L1 antibodies

How can researchers accurately measure ATG16L1-mediated autophagy induction?

For precise autophagy quantification using ATG16L1 antibodies, researchers should consider these methodological approaches:

Phospho-ATG16L1 Detection:
This represents an optimal approach as phospho-ATG16L1 is only present on newly forming autophagosomes, making it an excellent marker for autophagy induction . Quantification can be performed by:

• Western blot densitometry of phospho-ATG16L1 bands

• Counting phospho-ATG16L1-positive puncta in immunofluorescence images

• Comparing signals across experimental conditions while normalizing to appropriate controls

Co-localization Analysis:

• Perform double immunostaining for ATG16L1 and other autophagosome markers (e.g., LC3)

• Quantify co-localization using appropriate image analysis software

• Calculate Pearson's correlation coefficient or Manders' overlap coefficient

Autophagy Flux Assessment:

• Compare ATG16L1 localization patterns with and without lysosomal inhibitors

• Monitor changes in phospho-ATG16L1 levels in response to autophagy inducers

• Correlate findings with other autophagy markers for comprehensive analysis

How can ATG16L1 antibodies contribute to understanding membrane binding properties?

ATG16L1 contains an amphipathic α-helix (amino acids 113-131) that mediates membrane binding in vivo . Researchers can leverage ATG16L1 antibodies to study this interaction through:

Subcellular Fractionation Studies:

• Separate cellular components into cytosolic, membrane, and organelle fractions

• Detect ATG16L1 distribution across fractions using western blotting

• Compare distribution under basal conditions versus autophagy induction

• Quantify membrane association through densitometric analysis

Immunofluorescence Microscopy:

• Perform co-localization studies with membrane markers

• Analyze recruitment dynamics during autophagosome formation

• Compare wild-type ATG16L1 with membrane-binding domain mutants

• Quantify membrane association using high-resolution microscopy techniques

Structure-Function Analysis:

• Generate mutant constructs altering the amphipathic helix region

• Detect changes in localization and function using ATG16L1 antibodies

• Correlate membrane binding with autophagosome formation efficiency

What strategies can be employed to study ATG16L1 protein-protein interactions?

ATG16L1 forms complexes with multiple proteins during autophagosome formation. These interactions can be studied using:

Co-immunoprecipitation (Co-IP):

• Use ATG16L1 antibodies (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate) to pull down protein complexes

• Identify interacting proteins by western blotting or mass spectrometry

• Perform reverse Co-IP using antibodies against suspected binding partners

• Compare interaction profiles under different autophagy conditions

Proximity Ligation Assay (PLA):

• Utilize antibodies targeting ATG16L1 and potential interacting proteins

• Detect protein proximity through fluorescent signal generation

• Quantify interactions with subcellular resolution

• Map interaction networks during autophagosome formation

Domain Mapping Studies:

• Generate truncated ATG16L1 constructs lacking specific domains

• Use antibodies to detect changes in protein interaction patterns

• Identify critical regions for specific protein-protein interactions

• Correlate structural features with functional outcomes

How can ATG16L1 antibodies be applied in disease-related autophagy research?

Autophagy dysregulation has been implicated in numerous diseases. ATG16L1 antibodies can provide valuable insights through:

Expression Analysis in Disease Tissues:

• Compare ATG16L1 levels and localization in normal versus diseased tissues

• Correlate expression patterns with disease progression

• Identify alterations in specific isoform expression

• Develop potential diagnostic or prognostic markers

Genetic Variant Studies:

• Detect how disease-associated ATG16L1 variants affect protein expression and function

• Analyze variant-specific changes in protein stability or interactions

• Correlate genotype with cellular phenotypes and disease manifestations

Therapeutic Response Monitoring:

• Track changes in ATG16L1 expression/phosphorylation after autophagy-modulating treatments

• Correlate ATG16L1 status with treatment efficacy

• Identify potential predictive biomarkers for treatment response

How are new antibody technologies enhancing ATG16L1 research?

Recent technological advances are expanding the capabilities of ATG16L1 antibodies:

Super-Resolution Microscopy Applications:

• Use highly specific ATG16L1 antibodies with super-resolution techniques

• Map the precise localization of ATG16L1 during autophagosome formation

• Resolve interactions with other autophagy proteins at nanometer resolution

• Track dynamic changes in ATG16L1 distribution during autophagy progression

Multiplexed Detection Systems:

• Combine ATG16L1 antibodies with other autophagy markers in multiplexed assays

• Simultaneously detect multiple autophagy-related proteins in single samples

• Analyze autophagy pathway activation comprehensively

• Correlate ATG16L1 function with other autophagy components

Phospho-ATG16L1 Antibodies:
The development of phospho-specific ATG16L1 antibodies has created exciting new possibilities for studying autophagy induction, as they can detect endogenous phosphorylated ATG16L1 on newly forming autophagosomes without being affected by prolonged stress or late-stage autophagy blocks .

What methodological considerations apply when using ATG16L1 antibodies in advanced cell models?

As research moves toward more complex and physiologically relevant models, special considerations include:

3D Organoid Applications:

• Optimize fixation and permeabilization for tissue penetration

• Adjust antibody concentration and incubation times for 3D structures

• Use clearing techniques for deep tissue imaging

• Implement whole-mount staining protocols for comprehensive analysis

Live-Cell Imaging Compatibility:

• Combine ATG16L1 antibody fragment-based probes with live-cell techniques

• Monitor dynamic ATG16L1 redistribution during autophagy induction

• Correlate with functional autophagy outcomes

• Integrate with other live-cell autophagy markers

Patient-Derived Models:

• Validate antibody performance in human-derived samples

• Optimize protocols for limited clinical material

• Develop standardized quantification methods for translational research

• Correlate findings with patient clinical data