Overview of ATG4D and Its Role in Autophagy

ATG4D (Autophagy-Related Protein 4D) is a member of the ATG4 family of cysteine proteases, which are essential regulators of macroautophagy in mammalian cells. The ATG4 family in humans comprises four paralogs—ATG4A, ATG4B, ATG4C, and ATG4D—each capable of processing ATG8-family proteins (including LC3 and GABARAP subfamilies) through proteolytic cleavage of a C-terminal extension, thereby facilitating their lipidation and incorporation into the autophagosomal membrane.

ATG4D exhibits substrate specificity toward certain GABARAP-family members and has been implicated in mitophagy and selective autophagy pathways. Dysregulation of ATG4D activity has been associated with cellular stress responses, and research interest in this protease has grown in the context of cancer biology, neurodegeneration, and metabolic disorders. Recombinant Human ATG4D Protein (rp372233) provides researchers with a well-defined, recombinantly produced reagent to interrogate these functions in vitro.

Product Specifications and Quality Attributes

Recombinant Human ATG4D Protein (SKU: rp372233) is produced recombinantly to support consistent and reproducible experimental outcomes. Researchers should consult the product datasheet for lot-specific details including purity grade, host expression system, molecular weight, endotoxin level, and formulation buffer. General quality attributes typical of research-grade recombinant proteins from Aladdin Scientific include:

• Purity: Verified by SDS-PAGE and/or HPLC to confirm expected molecular weight and purity level (consult datasheet for lot-specific values).
• Endotoxin: Tested and specified per lot to meet requirements for cell-based assays.
• Formulation: Typically supplied lyophilized or in a stabilizing buffer; see product datasheet for exact formulation details.
• Biological Activity: Where applicable, activity is confirmed using a substrate cleavage or functional assay relevant to ATG4D protease function.

Always reference the current Certificate of Analysis (CoA) and product datasheet available on the Aladdin Scientific website for the most accurate and lot-specific specifications.

Reconstitution and Storage Protocol

Proper reconstitution and storage are critical for maintaining the activity and integrity of Recombinant Human ATG4D Protein. Follow these general guidelines, and always cross-reference the specific instructions provided in the product datasheet:

1. Reconstitution: If supplied in lyophilized form, briefly centrifuge the vial before opening to collect contents at the bottom. Reconstitute in the volume and buffer specified on the datasheet (typically sterile deionized water or a low-salt buffer supplemented with a carrier protein such as 0.1% BSA to enhance stability).
2. Mixing: Gently pipette to mix; do not vortex, as this may denature the protein.
3. Aliquoting: Immediately prepare single-use aliquots to minimize freeze-thaw cycles, which can reduce activity and increase aggregation.
4. Short-term storage: Store reconstituted protein at 4°C for up to 1 week if used frequently.
5. Long-term storage: Store aliquots at -20°C or -80°C. Avoid repeated freeze-thaw cycles (limit to ≤3 cycles as a general guideline).
6. Working concentration: Determine empirically for each assay; refer to literature precedents for ATG4 protease activity assays as a starting point.

Note: Specific buffer composition, reconstitution volume, and recommended working concentrations are provided on the product datasheet and CoA. Always use the datasheet as the primary reference.

Experimental Applications and Usage Recommendations

Recombinant Human ATG4D Protein is suitable for a range of in vitro biochemical and cell-free applications in autophagy research. Recommended and common applications include:

• In vitro protease activity assays: ATG4D can be used to assess cleavage of ATG8-family substrates (e.g., GABARAP, GABARAPL1, GABARAPL2/GATE-16) using fluorogenic peptide substrates or recombinant full-length ATG8 proteins as substrates.
• Protein–protein interaction studies: Co-immunoprecipitation, pull-down assays, or surface plasmon resonance (SPR) experiments to map binding partners relevant to the autophagy machinery.
• Structural and biophysical studies: ATG4D recombinant protein can serve as a reference standard or direct subject for X-ray crystallography, cryo-EM, or circular dichroism (CD) studies.
• Antibody validation: Use as a positive control antigen to validate ATG4D-specific antibodies in Western blot, ELISA, or immunofluorescence experiments.
• Drug discovery and screening: Suitable as a target protein in inhibitor or activator screening campaigns targeting the ATG4 protease family.

Researchers are advised to optimize assay conditions including pH, reducing agent concentration (e.g., DTT or TCEP are often included to maintain cysteine protease activity), temperature, and reaction time empirically. Refer to published ATG4 assay protocols in the literature for validated starting conditions.

Handling Precautions and Quality Assurance

To ensure optimal performance of Recombinant Human ATG4D Protein in downstream experiments, observe the following precautions:

• Protein handling: Always work on ice or in a cold environment when handling the reconstituted protein to minimize degradation.
• Contamination prevention: Use sterile, low-binding microtubes and pipette tips to prevent adsorption to surfaces and microbial contamination.
• Reducing conditions: As a cysteine protease, ATG4D activity is sensitive to oxidation. Maintain reducing conditions in assay buffers (e.g., 1–5 mM DTT or 0.5–1 mM TCEP) unless experimental design requires otherwise.
• Avoid repeated freeze-thaw: Pre-aliquot upon first use. Repeated freeze-thaw cycles degrade protein integrity and reduce enzymatic activity.
• Quality checks: Upon receipt, briefly centrifuge and visually inspect for solubility. For critical experiments, confirm activity using a pilot assay before committing the full preparation.
• Research use only: This product is intended for research purposes only and is not approved for diagnostic, therapeutic, or in vivo use.

For lot-specific quality data, download the Certificate of Analysis from the Aladdin Scientific product page for rp372233.