The ATG16 Antibody is a specialized immunological tool targeting the ATG16L1 protein, a critical component of the autophagy machinery. Autophagy-related 16-like 1 (ATG16L1) forms a complex with ATG5 and ATG12, essential for autophagosome formation—a key step in cellular degradation and recycling processes . This antibody is widely used to study autophagy mechanisms, inflammatory diseases (e.g., Crohn’s disease), and microbial infection responses .
ATG16 antibodies have been pivotal in elucidating autophagy-related pathways and disease mechanisms:
ATG16L1 Complex Formation: Antibodies confirmed ATG16L1’s role in the ~800 kDa ATG12-ATG5-ATG16L1 complex, critical for LC3 lipidation and autophagosome elongation .
Knock-out Models: Studies in ATG16L1−/− mice revealed defective autophagosome formation, elevated ubiquitinated proteins, and inflammatory cytokine upregulation .
Crohn’s Disease Link: The T300A polymorphism in ATG16L1 reduces autophagy efficiency and disrupts Paneth cell function, as shown via antibody-based protein detection in intestinal tissues .
T-cell Homeostasis: ATG16L1 deficiency in T cells leads to spontaneous intestinal inflammation and dysregulated Foxp3+ Treg cell survival, demonstrated through flow cytometry and WB .
Pathogen Clearance: ATG16 antibodies identified ATG16L1’s role in targeting Salmonella and Listeria via xenophagy, with WD40 domains essential for membrane repair during infection .
Biomarker Potential: Reduced ATG16L2 (a homolog) mRNA levels correlate with multiple sclerosis progression, highlighting cross-reactive antibody utility .
HIV-1 Survival: The rs6861 polymorphism in ATG16L1 enhances autophagy, delaying HIV-1 progression by improving T-cell effector responses .
While ATG16 antibodies are indispensable, challenges include cross-reactivity with homologs (e.g., ATG16L2) and batch variability. Emerging studies focus on isoform-specific antibodies and in vivo imaging applications .
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.
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 .
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 .
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
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
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
For diluted working solutions, prepare fresh or store short-term at 4°C with preservatives
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
Antibodies targeting AA 84-114, 11-257, or 501-607 for other domain-specific analyses
Species Reactivity:
Some antibodies react only with human samples
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
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
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
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
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
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
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 .
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