ATG4A Antibody

Basic Research Questions

How do I validate the specificity of ATG4A antibodies in Western blotting?

Validation requires a multi-step approach:

• Knockout controls: Use CRISPR-edited ATG4A-deficient cell lines (e.g., HAP1 or HeLa ATG4B/ATG4A double-KO models) to confirm the absence of non-specific bands .

• Isoform cross-reactivity tests: Compare antibody performance against recombinant ATG4A, ATG4B, ATG4C, and ATG4D proteins. For example, anti-ATG4A antibodies from R&D Systems (MAB4324) show no cross-reactivity with ATG4B in RAW 264.7 mouse cells .

• Tissue specificity validation: Test lysates from tissues with known ATG4A expression (e.g., human skeletal muscle) versus negative controls (e.g., liver) .

Example validation data table:

What experimental conditions optimize ATG4A detection in immunofluorescence?

Critical parameters derived from published protocols :

• Fixation: Ice-cold methanol (10 min) outperforms paraformaldehyde for cytoplasmic ATG4A preservation.

• Permeabilization: 0.2% Triton X-100 for 15 min balances membrane integrity and antibody access.

• Blocking: 5% BSA + 10% normal goat serum reduces non-specific binding in neuronal cultures.

• Antibody concentration: Titrate between 10-50 µg/mL; 25 µg/mL shown effective in RAW 264.7 macrophages .

Pro Tip: Combine with lysosomal inhibitors (e.g., 100 nM bafilomycin A1 for 4 hr) to amplify autophagosome-associated ATG4A signals .

How should researchers handle discrepancies between ATG4A mRNA and protein expression data?

Methodological solutions:

• Post-translational regulation assessment:

  • Treat cells with 10 µM MG-132 (proteasome inhibitor) for 6 hr to detect ubiquitination

  • Perform cycloheximide chase assays (100 µg/mL, 0-8 hr time course)

• Antibody validation:

  • Compare multiple clones (e.g., MAB4324 vs AF4324) across cell lines

  • Use RNAscope® ISH as orthogonal validation in parallel

Advanced Research Questions

How to design experiments resolving ATG4A's dual roles in autophagy initiation vs. termination?

Strategies from cutting-edge studies :

• Stage-specific inhibition:

  • Initiation: Transfect cells with ATG4A(C74A) dominant-negative mutant

  • Termination: Use LC3B-II-specific nanobodies to block delipidation

• Temporal analysis:

  • Combine live-cell imaging (mCherry-ATG4A) with rapamycin/bafilomycin time courses

  • Quantify LC3B-II/I ratios at 15-min intervals for 6 hr post-induction

Key finding from :

What methods address contradictory findings about ATG4A's role in apoptosis?

Three-pronged approach based on :

• Context-dependent analysis:

  • Test in H2O2-induced oxidative stress (50-200 µM, 24 hr) vs. serum starvation models

  • Measure caspase-3 cleavage alongside LC3 lipidation

• Isoform compensation assays:

  • Use siRNA pools targeting ATG4A/B/C/D in combination

  • Employ isoform-specific antibodies (e.g., MAB4324 for ATG4A, ABF124 for ATG4B)

• Spatial resolution:

  • Perform subcellular fractionation followed by immunoprecipitation-MS

  • Correlate mitochondrial vs. ER-localized ATG4A with apoptosis markers

How to investigate ATG4A's non-autophagic functions in protein secretion?

Novel methodology from recent work:

• Proteomic profiling:

  • SILAC labeling + ATG4A immunoprecipitation in GRASP55 KO cells

  • Identify cargo proteins using TIMS-TOF MS (0.5% FDR threshold)

• Live-cell trafficking assays:

  • Microfluidics-based secretion chambers with ATG4A pHluorin reporters

  • Quantify ER-to-Golgi transit times under nutrient stress

What computational tools predict ATG4A-substrate interactions?

Integrated pipeline validated in :

• Molecular docking:

  • Use HADDOCK 2.4 with ATG4A crystal structure (PDB 4RNG)

  • Screen against LC3/GABARAP family members

• MD simulations:

  • 100 ns runs in GROMACS 2022 with CHARMM36m force field

  • Analyze binding free energies (MM/PBSA) for mutation hotspots

• Evolutionary analysis:

  • Apply ConSurf to identify conserved residues across 47 vertebrate species

Methodological Best Practices

• Antibody lot validation: Always repeat key experiments with new antibody batches using standardized lysates (e.g., ATG4A-transfected HEK293T cells)

• Multiplexed autophagy flux assays: Combine ATG4A staining with LysoTracker Red (100 nM, 30 min) and LC3B-I/II immunoblotting

• Data normalization: Use total protein staining (e.g., REVERT™) instead of housekeepers for quantitative Westerns

Ethical Note: For animal studies using ATG4A antibodies, confirm IACUC approval for species cross-reactivity (e.g., mouse vs human ATG4A shares 94% identity ).