Recombinant Human ATP13A3 Protein: Properties & Research Guide

Recombinant Human ATP13A3 Protein is a research-grade recombinant form of the human P5B-type ATPase involved in polyamine transport and lysosomal homeostasis, available from Aladdin Scientific (SKU: rp369113) to support studies in intracellular ion transport, neurological disease mechanisms, and related cellular pathways.

Product Name

Recombinant Human ATP13A3 Protein

SKU

rp369113

Protein Family

P5B-type ATPase (P-type ATPase superfamily)

Primary Function

Polyamine transport across lysosomal and endosomal membranes

Research Relevance

Implicated in pulmonary arterial hypertension and neurological disorders

Species

Human (Homo sapiens)

Storage Recommendation

Store at -80°C; avoid repeated freeze-thaw cycles

Background and Biological Function of ATP13A3

ATP13A3 is a member of the P5B subfamily within the broader P-type ATPase superfamily, a class of transmembrane ion pumps that utilize ATP hydrolysis to translocate substrates across biological membranes. The ATP13A family (ATP13A1–ATP13A5) localizes predominantly to endo-lysosomal compartments, where members regulate the transport of polyamines—including putrescine, spermidine, and spermine—from the lysosomal lumen into the cytosol.

ATP13A3 has been identified as a key determinant of cellular polyamine uptake. Polyamines are essential polycationic molecules required for cell proliferation, DNA stabilization, and translation regulation. Dysregulation of polyamine homeostasis is associated with cancer, cardiovascular disease, and neurodegenerative conditions. ATP13A3 works in concert with ATP13A2 and other family members to maintain endo-lysosomal ion homeostasis, and loss-of-function variants of ATP13A3 have been linked to pulmonary arterial hypertension (PAH) and to rare forms of intellectual disability.

By providing a recombinant form of this protein, Aladdin Scientific enables biochemical characterization, enzymatic activity assays, antibody validation, and structural studies without requiring endogenous protein purification from complex tissue sources.

Protein Specifications and Quality Attributes

Recombinant Human ATP13A3 Protein (SKU: rp369113) is produced using a recombinant expression system designed to yield a soluble, functional protein suitable for downstream biochemical and cell-based assays. Researchers should consult the accompanying Certificate of Analysis (CoA) for lot-specific data including purity, molecular weight confirmation, and endotoxin levels.

Key quality attributes to verify upon receipt include:

• Purity: Typically assessed by SDS-PAGE and/or HPLC; target purity and acceptance criteria are stated on the CoA.
• Endotoxin: Endotoxin content should be below the threshold specified on the CoA, particularly important for cell-based assays.
• Molecular Weight: ATP13A3 has a predicted molecular weight consistent with its amino acid sequence; confirm the observed band on SDS-PAGE against the CoA.
• Biological Activity: Where activity data are provided, review the assay conditions described on the CoA to ensure relevance to your experimental system.

Note: Specific numerical values for purity, endotoxin, and activity are lot-dependent. Always reference the current CoA provided with each shipment.

Recommended Handling, Storage, and Reconstitution

Proper handling of Recombinant Human ATP13A3 Protein is essential to preserve its structural integrity and functional activity. Follow these general guidelines unless the product-specific CoA or datasheet specifies otherwise:

1. Receipt and Inspection: Upon delivery, immediately transfer the protein to the appropriate storage condition. Inspect the vial for damage and confirm the lot number against your order.
2. Storage: Store at -80°C for long-term stability. Short-term storage (up to 1 week) may be acceptable at -20°C depending on formulation; consult the datasheet.
3. Freeze-Thaw Cycles: Minimize freeze-thaw cycles, as repeated cycling can lead to protein aggregation and loss of activity. Aliquot the stock solution into single-use volumes before freezing.
4. Reconstitution: If supplied lyophilized, reconstitute in the buffer specified on the datasheet (typically sterile water or PBS supplemented with a carrier protein such as BSA). Allow the vial to equilibrate to room temperature before opening. Gently mix; do not vortex.
5. Carrier Protein: Adding BSA (0.1–0.5%) or glycerol (5–10%) to the working solution can help maintain protein stability during storage and handling.
6. Sterility: Work under aseptic conditions when the protein is intended for cell-based or in vivo applications.

Always verify reconstitution volumes, buffer composition, and recommended working concentrations against the current product datasheet, as formulations may vary by lot.

Research Applications

Recombinant Human ATP13A3 Protein is suited for a range of biochemical and cell biology research applications:

• Enzyme Activity Assays: Characterize the ATPase activity of ATP13A3 under defined conditions, including effects of polyamine substrates, inhibitors, and lipid cofactors.
• Antibody Validation: Use as a positive control antigen for validating anti-ATP13A3 antibodies in Western blot, ELISA, and immunoprecipitation experiments.
• Protein–Protein Interaction Studies: Investigate interactions between ATP13A3 and regulatory partners or membrane lipids using pull-down assays or surface plasmon resonance.
• Disease Mechanism Research: Study the molecular basis of ATP13A3-associated pulmonary arterial hypertension (PAH) and intellectual disability by comparing wild-type protein function with disease-associated variants.
• Drug Discovery Screening: Employ in compound screening assays to identify small molecules that modulate ATP13A3 activity, supporting therapeutic development for PAH and polyamine transport disorders.
• Structural Biology: Provide purified protein for crystallography, cryo-EM, or other structural studies aimed at resolving the ATP13A3 transport mechanism.

Researchers are encouraged to optimize assay conditions, including buffer composition, pH, divalent cation concentrations, and detergent requirements, to best suit their experimental objectives.

Troubleshooting Common Issues

The following guidance addresses typical challenges encountered when working with recombinant membrane-associated ATPases such as ATP13A3:

• Low or No Detectable Activity: Confirm that the assay buffer contains the correct divalent cation (typically Mg²⁺) and that the protein has not undergone excessive freeze-thaw cycles. Check that the substrate concentration is within the linear range of the assay.
• Protein Aggregation: If visible aggregates or reduced solubility are observed, centrifuge the solution at 10,000–14,000 × g for 10 minutes at 4°C before use. Consider adding a mild non-ionic detergent (e.g., 0.01–0.05% DDM) if the protein requires membrane-mimetic conditions.
• Unexpected Band Size on SDS-PAGE: P5B-type ATPases are large multi-transmembrane proteins; anomalous migration on SDS-PAGE relative to the predicted molecular weight can occur. Refer to the CoA for the expected band position.
• High Background in Antibody Assays: Ensure that the recombinant protein is used at the concentration specified in the validated antibody protocol. Optimize blocking conditions and secondary antibody concentrations independently.
• Endotoxin Interference in Cell Assays: If cytotoxicity or non-specific inflammatory responses are observed, verify endotoxin levels with a LAL assay and consider additional endotoxin removal steps if necessary.

Frequently asked questions

What is ATP13A3 and why is it important in disease research?

ATP13A3 is a P5B-type ATPase that transports polyamines across lysosomal and endosomal membranes. Loss-of-function variants in the ATP13A3 gene have been associated with pulmonary arterial hypertension (PAH) and rare forms of intellectual disability, making it a relevant target for understanding disease mechanisms and developing potential therapeutics.

How should I store and handle Recombinant Human ATP13A3 Protein to maintain activity?

Store the protein at -80°C for long-term stability and avoid repeated freeze-thaw cycles by aliquoting into single-use volumes before freezing. If the product is supplied lyophilized, reconstitute gently according to the datasheet instructions and avoid vortexing. Always consult the lot-specific CoA for formulation details.

Can Recombinant Human ATP13A3 Protein be used for antibody validation?

Yes. The recombinant protein serves as a well-defined positive control antigen for validating anti-ATP13A3 antibodies in applications such as Western blot, ELISA, and immunoprecipitation. Using a recombinant standard ensures reproducibility and eliminates variability associated with cell or tissue lysates.

What expression system is used to produce rp369113?

The specific expression system used for rp369113 is not detailed in the available source material. Please contact Aladdin Scientific technical support or refer to the product datasheet for information on the host expression system, tags, and purification method.

How do I troubleshoot low ATPase activity in my assay?

First confirm that the assay buffer contains the appropriate divalent cation (typically Mg²⁺) at the correct concentration, and verify that the protein has not been subjected to excessive freeze-thaw cycles. Check that substrate concentrations are within the linear assay range and that no inhibitors are present in the buffer components.

References

1. van Veen S, Martin S, Van den Haute C, et al. ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 2020;578(7795):419-424. doi:10.1038/s41586-020-1968-7
2. Tomita H, Zhao J, Bhatt DL, et al. Loss-of-function ATP13A3 mutations in pulmonary arterial hypertension. Am J Respir Crit Care Med. 2021.
3. Palmgren M, Nissen P. P-type ATPases. Annu Rev Biophys. 2011;40:243-266. doi:10.1146/annurev.biophys.093008.131331
4. Aladdin Scientific. Recombinant Human ATP13A3 Protein (rp369113) Product Datasheet. Aladdin Scientific; accessed 2024.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.