ATG4D Antibody

What is ATG4D and why is it important in research?

ATG4D (Autophagy related 4D Cysteine Peptidase) is one of four ATG4 cysteine proteases (ATG4A-D) that play crucial roles in autophagy regulation through the priming and deconjugation of Atg8-family proteins. ATG4D has gained increasing research interest due to its recently discovered links to neurological disorders. Bi-allelic variants in the ATG4D gene have been associated with a neurodevelopmental disorder characterized by speech and motor impairment . ATG4D is particularly important because it demonstrates dual functionality - it plays roles in both autophagy-dependent and autophagy-independent cellular processes, including mitochondrial function and apoptosis regulation .

What types of ATG4D antibodies are available for research?

Various ATG4D antibodies are available targeting different epitopes of the protein:

This diversity allows researchers to select antibodies specifically targeting regions of interest, particularly when studying ATG4D variants or processing events like caspase cleavage .

What applications can ATG4D antibodies be used for?

ATG4D antibodies can be applied in multiple experimental techniques:

• Western Blotting (WB): For detection of ATG4D protein (expected MW ~53 kDa) and processed forms (~47 kDa for caspase-cleaved, ~42 kDa for mitochondrial form)

• Immunohistochemistry (IHC): For tissue localization studies, including paraffin-embedded sections

• Immunofluorescence (IF): For subcellular localization studies, particularly useful for examining mitochondrial targeting

• ELISA: For quantitative detection of ATG4D in various sample types

• Cytometric bead array: When using matched antibody pairs

The selection of appropriate applications depends on experimental goals, with consideration for specific antibody validation data provided by manufacturers .

How should ATG4D antibodies be stored and handled?

Optimal storage conditions for ATG4D antibodies typically include:

• Long-term storage: -20°C to -80°C (antibody-dependent)

• Short-term storage: 2-8°C for up to two weeks

• Buffer composition: Most are supplied in PBS with stabilizers such as glycerol (50%) and preservatives like sodium azide (0.02-0.09%)

• Avoid repeated freeze-thaw cycles: Aliquoting is recommended for antibodies stored at -20°C

For conjugation-ready formats (BSA and azide-free), special handling may be required, particularly maintaining sterile conditions and using within recommended timeframes .

How can I validate the specificity of my ATG4D antibody?

Comprehensive validation requires multiple approaches:

• Positive and negative controls:

  • Positive: Human brain tissue shows reliable detection of ATG4D at 53 kDa

  • Negative: Use ATG4D knockout cell lines (e.g., ATG4 tetra knockout models) to confirm specificity

• Knockdown validation:

  • Implement siRNA or shRNA against ATG4D and demonstrate reduction in detected signal

  • Published studies utilizing KD/KO validation can provide reference protocols

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (when available) before application

  • Signal should be significantly reduced if antibody is specific

• Cross-reactivity assessment:

  • Test against other ATG4 family members (ATG4A, ATG4B, ATG4C)

  • Some antibodies are specifically designed to not cross-react with other ATG4 proteins

A comprehensive validation approach increases confidence in experimental findings and should be documented in research publications .

What are the optimal sample preparation methods for detecting ATG4D?

Sample preparation should be tailored to the specific application and cellular compartment of interest:

• For total cellular ATG4D (Western blotting):

  • Use RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Load 30-35 μg of total protein per lane (based on successful detection in HL-60 cells)

• For detecting mitochondrial ATG4D:

  • Implement subcellular fractionation to isolate mitochondria

  • Mitochondrial ATG4D is processed to ~42 kDa form, distinct from the full-length 53 kDa protein

  • Include both cytosolic and mitochondrial fractions for comparison

• For tissue sections (IHC/IF):

  • Paraffin-embedded sections require antigen retrieval (typically heat-induced in citrate buffer)

  • Fresh-frozen sections may provide better antigen preservation for some epitopes

  • Use chloroquine (50 μM, 16h) treatment of cells for enhanced autophagy-related detection

Optimization of sample preparation is critical for successful ATG4D detection across different experimental contexts .

How can I design experiments to study caspase-cleaved ATG4D?

Caspase-cleaved ATG4D (ΔN63 ATG4D) has distinct properties from the full-length protein and requires specific experimental approaches:

• Induction of caspase cleavage:

  • Treat cells with apoptosis inducers like staurosporine

  • Monitor PARP cleavage (p85) as a parallel marker of caspase activation

  • Full ATG4D diminishes around 8 hours post-treatment with complete disappearance by 24 hours

• Mutation of caspase cleavage site:

  • Create DEVA⁶³K mutant (D63A) as a non-cleavable control

  • Compare with wild-type ATG4D in apoptosis assays

• Subcellular localization studies:

  • Use fluorescently tagged constructs to track localization

  • Compare full-length ATG4D (cytosolic) with ΔN63 ATG4D (mitochondrial)

  • Note that N-terminal tagging of ΔN63 ATG4D blocks mitochondrial localization

• Functional assessment:

  • Measure GABARAP-L1 processing activity in vitro

  • ΔN63 ATG4D shows enhanced activity against LC3B, GABARAPL1, and GABARAPL2 compared to full-length protein

A comprehensive experimental design incorporates both localization and functional assessments to fully characterize this important processed form of ATG4D .

How do ATG4D mutations affect autophagy and neurological function?

Recent research has identified bi-allelic ATG4D variants associated with neurodevelopmental disorders, providing insights into mutation effects:

• Functional impact assessment:

  • GABARAPL1 priming assay shows decreased activity for three of four identified patient variants

  • Rescue experiments in ATG4 tetra knockout cells confirm functional impairment of missense variants in the cysteine protease domain

• Autophagy pathway analysis:

  • Autophagosome biogenesis and induction appear intact in cells with ATG4D variants

  • Specific defects occur in GABARAPL1 processing, suggesting selective rather than global autophagy disruption

• Protein expression effects:

  • Patient-derived variants did not significantly alter ATG4D mRNA expression or protein levels

  • Primary impact appears to be on protein functionality rather than expression or stability

• Experimental approaches for neurological studies:

  • Patient-derived fibroblasts and lymphoblastoid cell lines provide disease-relevant models

  • Animal models (zebrafish, mouse) support ATG4D's neuroprotective role

The emerging data suggests that ATG4D mutations contribute to neurological pathology through specific functional deficits rather than complete loss of autophagy capacity .

What experimental controls are critical when studying ATG4D in autophagy research?

Robust autophagy research requires careful controls specific to ATG4D studies:

• ATG4 paralog controls:

  • Include analysis of other ATG4 family members (ATG4A, ATG4B, ATG4C) to assess compensatory effects

  • ATG4B is the dominant ATG4 family member, with broader substrate specificity than ATG4D

• Autophagy flux controls:

  • Incorporate bafilomycin A1 treatment to distinguish changes in autophagy initiation versus flux

  • Monitor both LC3-I to LC3-II conversion and p62/SQSTM1 degradation

• Substrate specificity controls:

  • ATG4D preferentially processes GABARAPL1, but also acts on LC3B and GABARAPL2

  • Include multiple ATG8 family proteins in functional assays

• Mitochondrial function controls:

  • For studies of mitochondrial ATG4D, include measures of mitochondrial membrane potential

  • CCCP treatment provides a useful experimental condition that specifically reveals mitochondrial ATG4D's role in cell death sensitization

• Experimental validation table:

How can ATG4D antibodies be used to investigate extracellular vesicle (EV) release?

ATG4D has emerging roles in extracellular vesicle biology that can be investigated using specific protocols:

• EV isolation and characterization:

  • Differential ultracentrifugation followed by density gradient purification

  • Nanoparticle tracking analysis for size distribution and concentration

  • Western blotting using ATG4D antibodies to detect protein in EV fractions

• Proteomics approach:

  • Quantitative proteomic analysis comparing EVs from control and ATG4D mutant cells

  • Assessment of peptide spectrum matches (PSM) values for EV markers

  • Monitoring of EV-associated proteins like integrins (ITGA1, ITGA5, ITGB1), heparan sulfate proteoglycans, CD44, and heat-shock proteins (HSPA8, HSP90AB1)

• Purity assessment methods:

  • Monitor non-EV protein contamination (Category 3 proteins per Théry et al.)

  • Compare APOA, APOB, and albumin levels between sample types

  • Ensure PSM values for contaminants are lower than for genuine EV markers

• Experimental validation of ATG4D effects:

  • Compare EV release from wild-type, ATG4D knockout, and ATG4D mutant cells

  • Assess changes in EV cargo composition using antibody arrays or proteomics

  • Evaluate functional impacts through EV transfer experiments

This emerging research area connects ATG4D function to intercellular communication mechanisms beyond its canonical autophagy role .

What are the methodological considerations for studying mitochondrial ATG4D?

The mitochondrial localization of caspase-cleaved ATG4D requires specialized experimental approaches:

• Localization verification methods:

  • Mitochondrial fractionation followed by Western blotting

  • Confocal microscopy with mitochondrial markers (e.g., MitoTracker)

  • Super-resolution techniques for detailed intramitochondrial distribution

• Mitochondrial targeting sequence analysis:

  • The cryptic mitochondrial targeting sequence is exposed upon caspase cleavage

  • Using deletion constructs can help map essential targeting elements

  • The sequence 64-105 of ATG4D effectively targets GFP to mitochondria

• Functional assessment protocols:

  • Measure mitochondrial reactive oxygen species (ROS) with specific probes

  • Analyze cristae organization using transmission electron microscopy

  • Assess mitochondrial membrane potential with JC-1 or TMRE dyes

• Disease model applications:

  • Erythropoiesis models show altered mitochondrial clearance with ΔN63 ATG4D expression

  • Cell death assays reveal sensitization to CCCP but not other mitochondrial toxins

  • Distinguishing between autophagic and non-autophagic effects requires careful experimental design

• Experimental workflow:

This methodological framework enables comprehensive investigation of this unique aspect of ATG4D biology .

How can I address weak or non-specific signals when using ATG4D antibodies?

Optimizing signal detection requires systematic troubleshooting:

• Sample-related optimizations:

  • Increase protein loading (35-50 μg recommended for Western blotting)

  • Use fresh samples to avoid protein degradation

  • Consider tissue specificity (human brain tissue shows reliable detection)

• Antibody concentration adjustments:

  • Titrate antibody dilutions (typical range: 1:500-1:1000 for Western blotting)

  • Extended incubation times may improve weak signals (overnight at 4°C)

  • Signal enhancement systems may be beneficial for low abundance detection

• Buffer and blocking optimizations:

  • Test different blocking agents (BSA vs. non-fat milk)

  • Optimize incubation times and washing procedures

  • Consider specialized buffers for different applications

• Signal enhancement strategies:

  • For immunofluorescence, treatment with chloroquine (50 μM, 16h) enhances signal detection

  • Super-resolution microscopy may be required for detailed localization studies

  • Tyramide signal amplification for IHC with low abundance targets

Systematic optimization addressing each variable individually will help achieve optimal signal detection while maintaining specificity .

How should data contradictions between different ATG4D antibodies be interpreted?

Researchers may encounter conflicting results when using different ATG4D antibodies, requiring careful analytical approaches:

• Epitope-specific considerations:

  • Different antibodies target distinct regions (N-terminal, central, C-terminal)

  • The caspase-cleaved form (ΔN63 ATG4D) will not be detected by N-terminal antibodies

  • Some antibodies detect only specific isoforms

• Validation comparison:

  • Evaluate validation methods used for each antibody

  • Antibodies validated in knockout systems provide higher confidence

  • Consider published literature using specific antibody clones

• Resolution strategies:

  • Use multiple antibodies targeting different epitopes

  • Implement complementary techniques (e.g., mass spectrometry)

  • Consider genetic approaches (epitope tagging) for definitive identification

• Reporting recommendations:

  • Document complete antibody information (catalog number, lot, dilution)

  • Specify validation methods employed

  • Note limitations when interpreting results

Data contradictions often reveal biological complexities rather than technical failures and may provide insights into protein processing, isoform expression, or post-translational modifications .

What methodological adaptations are needed for studying ATG4D in different species?

Species-specific considerations are essential when working with ATG4D across different model organisms:

• Antibody cross-reactivity assessment:

  • Verify species reactivity (human, mouse, rat common; pig predicted)

  • Sequence alignment of epitope regions across species

  • Validation in species-specific positive controls

• Species-specific experimental design:

  • Mouse models: Several antibodies show confirmed reactivity

  • Rat models: Fewer validated antibodies (83951-4-PBS confirmed)

  • Non-mammalian models: May require custom antibody development

• Experimental design considerations:

  • Dog models: ATG4D mutations linked to neurodegenerative vacuolar storage disease

  • Zebrafish models: Support neuroprotective role of ATG4D

  • Cell line selection should match antibody species reactivity

• Cross-species comparison table:

Species-specific optimization ensures reliable results across different model systems used in ATG4D research .

How can ATG4D antibodies contribute to understanding neurodevelopmental disorders?

Emerging roles of ATG4D in neurological conditions open new research avenues:

• Patient variant screening approaches:

  • Develop antibodies specifically recognizing common mutations (p.Leu244Pro, p.Gly170Asp)

  • Implement high-throughput screening of patient samples

  • Correlate ATG4D function with clinical phenotypes

• Functional imaging methodologies:

  • Live-cell imaging of ATG4D dynamics in neuronal models

  • Correlate localization patterns with neuronal development and function

  • Track substrate processing in real-time using fluorescent reporters

• Therapeutic development applications:

  • Screen for compounds that enhance residual ATG4D activity

  • Develop assays measuring GABARAPL1 processing for drug screening

  • Create experimental platforms for gene therapy approaches

• Biomarker development potential:

  • Explore ATG4D activity as a diagnostic or prognostic marker

  • Investigate extracellular vesicle ATG4D content as accessible biomarker

  • Develop quantitative assays for clinical application

The growing connection between ATG4D and neurological conditions provides compelling opportunities for translational research using well-characterized antibodies .

What novel experimental designs could advance understanding of ATG4D's dual roles in autophagy and apoptosis?

Innovative approaches are needed to dissect ATG4D's complex biological functions:

• Proximity labeling techniques:

  • BioID or TurboID fusion with full-length versus ΔN63 ATG4D

  • Identify compartment-specific interaction partners

  • Map dynamic protein interactions during cellular stress

• Substrate specificity profiling:

  • Develop high-throughput assays for ATG4D activity against multiple substrates

  • Compare substrate preferences of full-length versus cleaved forms

  • Engineer substrate reporter systems for live-cell applications

• Conditional expression systems:

  • Create inducible models to temporally control ATG4D expression

  • Develop compartment-specific targeting strategies

  • Design genetic complementation systems in knockout backgrounds

• Integrated multi-omics approaches:

  • Combine proteomics, metabolomics, and transcriptomics

  • Map ATG4D-dependent pathways during different cellular states

  • Identify novel regulatory mechanisms and targets

These advanced experimental strategies will help clarify how ATG4D integrates autophagy regulation with mitochondrial function and cell death pathways .