Recombinant Human ATP13A1 Protein: Expression & Usage Protocol

Recombinant Human ATP13A1 Protein is a research-grade P5-type ATPase used to investigate lysosomal homeostasis, heavy-metal transport, and related cellular pathways in human biology.

Product Name

Recombinant Human ATP13A1 Protein

SKU

rp377698

Protein Family

P5-type ATPase (ATP13A family)

Species Origin

Human (Homo sapiens)

Primary Application

Biochemical assays, lysosomal function studies, ATPase activity assays

Storage Recommendation

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

Background & Biological Significance of ATP13A1

ATP13A1 is a member of the P5-type ATPase subfamily, a group of evolutionarily conserved transmembrane ion pumps. In humans, ATP13A1 is ubiquitously expressed and localizes predominantly to the endoplasmic reticulum and lysosomal membranes, where it is believed to regulate divalent cation homeostasis, including zinc and manganese transport. Disruption of ATP13A1 function has been associated with defects in protein quality control, mitochondrial function, and lysosomal integrity. Understanding ATP13A1 activity is relevant to research in neurodegeneration, heavy-metal toxicity, and cellular stress responses. Recombinant Human ATP13A1 Protein (rp377698) provides a defined, research-grade tool for dissecting these molecular functions in vitro.

Reagent Preparation & Reconstitution

Materials required: Recombinant Human ATP13A1 Protein (rp377698), sterile deionized water or compatible reconstitution buffer (e.g., PBS, pH 7.4, with 10% glycerol), microcentrifuge tubes, and ice.

Step 1 – Equilibration: Remove the vial from -80°C storage and allow it to equilibrate briefly on ice for 5–10 minutes. Do not allow the protein to warm to room temperature before reconstitution.

Step 2 – Reconstitution: Add the appropriate volume of sterile reconstitution buffer slowly to the lyophilized pellet. Gently swirl or pipette to mix; do not vortex, as vigorous agitation may denature the protein.

Step 3 – Clarification: Centrifuge briefly (1,000 × g, 30 seconds) to collect any undissolved material. Transfer the clear supernatant to a fresh microcentrifuge tube.

Note: Confirm specific reconstitution volume and buffer from the Certificate of Analysis (CoA) supplied with the product lot, as these parameters may vary by lot.

Experimental Applications & Assay Guidance

Recombinant Human ATP13A1 Protein (rp377698) is suitable for a range of in vitro biochemical applications:

• ATPase Activity Assay: Use colorimetric or fluorometric phosphate-release assays to measure hydrolytic activity. Include appropriate positive and negative controls; optimize MgCl₂ and ATP concentrations empirically for your system.
• Protein–Protein Interaction Studies: Employ pull-down, co-immunoprecipitation, or surface plasmon resonance (SPR) approaches to identify binding partners of ATP13A1.
• Structural Studies: The recombinant protein may serve as antigen for antibody generation or as a reference standard in western blot assays.
• Inhibitor Screening: Use the recombinant protein in high-throughput compatible ATPase inhibition assays to identify small-molecule modulators.

Always titrate protein concentration to determine optimal working amounts for each specific assay format. Refer to peer-reviewed literature for validated assay conditions relevant to P5-type ATPases.

Storage, Stability & Handling Precautions

Long-term storage: Store lyophilized protein at -80°C. Once reconstituted, aliquot into single-use volumes to prevent repeated freeze-thaw cycles, which can cause aggregation and loss of activity.

Short-term storage: Reconstituted protein may be stored at 4°C for up to 48–72 hours if used within that window. For extended storage after reconstitution, add a carrier protein such as 0.1% BSA and store at -80°C.

Handling: Work on ice at all times. Use low-binding microcentrifuge tubes to minimize adsorption losses. Wear appropriate PPE (gloves, lab coat, eye protection) when handling protein solutions.

Quality control: Verify integrity by SDS-PAGE or western blot prior to use in critical experiments. The expected molecular weight should correspond to the full-length or tagged construct as specified in the product CoA.

Note: Specific purity percentage, endotoxin level, and formulation details should be confirmed from the product CoA for lot rp377698. The above are general best-practice guidelines.

Troubleshooting

Problem: Low or no ATPase activity detected.
Possible causes: Protein aggregation due to improper reconstitution or freeze-thaw damage; incorrect buffer composition; suboptimal substrate concentrations. Solutions: Verify reconstitution procedure; centrifuge sample at 10,000 × g for 10 minutes to remove aggregates; optimize Mg²⁺/ATP ratio.

Problem: Multiple bands on SDS-PAGE.
Possible causes: Proteolytic degradation during handling; disulfide-linked aggregates. Solutions: Add protease inhibitors during reconstitution; include reducing agent (e.g., 1 mM DTT) in sample buffer where compatible.

Problem: High background in binding assay.
Possible causes: Non-specific adsorption of protein to assay surfaces. Solutions: Pre-block surfaces with BSA; use low-binding plates or tubes; optimize washing steps.

If issues persist, contact Aladdin Scientific technical support and provide your lot number (rp377698) and CoA details for tailored assistance.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human ATP13A1 Protein (rp377698)?

A standard reconstitution buffer such as sterile PBS (pH 7.4) supplemented with 10% glycerol is generally suitable for P5-type ATPase recombinant proteins to maintain solubility and stability. However, always refer to the Certificate of Analysis (CoA) supplied with your specific product lot (rp377698) for the manufacturer-recommended buffer and volume, as these may be lot-specific.

How should I store Recombinant Human ATP13A1 Protein to preserve activity?

Lyophilized protein should be stored at -80°C until use. After reconstitution, aliquot into single-use volumes immediately and store aliquots at -80°C with a carrier protein (e.g., 0.1% BSA) to minimize freeze-thaw degradation. Avoid storing reconstituted protein at 4°C for more than 48–72 hours.

What applications is Recombinant Human ATP13A1 Protein suitable for?

Recombinant Human ATP13A1 Protein (rp377698) is suitable for in vitro biochemical applications including ATPase activity assays, protein–protein interaction studies (pull-down, co-IP, SPR), use as an antigen for antibody production, western blot reference standards, and small-molecule inhibitor screening assays. Researchers should validate the protein's performance in their specific assay system.

How do I verify the quality of the recombinant protein before use?

Protein quality can be assessed by running an SDS-PAGE gel to confirm the expected molecular weight band and evaluate purity. Western blot analysis using an anti-ATP13A1 or anti-tag antibody can confirm protein identity. Endotoxin levels and purity specifications should be available on the product CoA.

Can Recombinant Human ATP13A1 Protein be used for cell-based assays?

Recombinant Human ATP13A1 Protein is primarily intended for in vitro biochemical assays. Use in cell-based assays would require additional considerations such as low endotoxin levels, appropriate delivery methods, and functional validation in the chosen cell system. Consult the CoA for endotoxin specifications and contact Aladdin Scientific technical support for further guidance.

References

1. Sørensen DM, Møller AB, Jakobsen MK, Jensen MK, Vangheluwe P, Buch-Pedersen MJ, Palmgren MG. (2012). Ca2+ induces pseudo-reversion of a Na+-specific P-type ATPase. J Biol Chem. 287(33):27609-27619.
2. van Veen S, Martin S, Van den Haute C, Benoy V, Lyons J, Vanhoutte R, Kahler JP, Decuypere JP, Gelinas R, De Roo E, et al. (2020). ATP13A2 deficiency disrupts lysosomal polyamine export. Nature. 578(7795):419-424.
3. Palmgren M, Østerberg JT, Bhati KK, Bhati M, Bhati R. (2020). Evolution of P-type ATPases. Front Plant Sci. (Review article on P-type ATPase family classification).

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