Recombinant Human ATP7B Protein: Research Guide & Protocol

Recombinant Human ATP7B Protein is a recombinant form of the human copper-transporting P-type ATPase encoded by the ATP7B gene, essential for hepatic copper homeostasis and widely used as a research reagent in studies of Wilson disease pathology, copper metabolism, and related therapeutic target validation.

Product Name

Recombinant Human ATP7B Protein

SKU

rp366473

Gene/Protein Target

ATP7B (ATPase Copper Transporting Beta)

Protein Family

P-type ATPase, copper transporter subfamily

Primary Research Application

Wilson disease research, copper homeostasis studies, enzyme activity assays

Biological Function

Intracellular copper trafficking and biliary copper excretion in hepatocytes

Biological Background of ATP7B

ATP7B encodes a P1B-type ATPase that plays a central role in copper homeostasis within the human body. It is predominantly expressed in the liver, where it performs two critical functions: incorporating copper into ceruloplasmin in the trans-Golgi network, and exporting excess copper into bile for elimination. Loss-of-function mutations in the ATP7B gene cause Wilson disease (OMIM #277900), an autosomal recessive disorder characterized by toxic copper accumulation in the liver, brain, and other organs.

The ATP7B protein contains eight transmembrane domains, six copper-binding CXXC motifs in its N-terminal domain, an ATP-binding domain, and an actuator domain, all features characteristic of heavy-metal P-type ATPases. Understanding ATP7B structure-function relationships is fundamental to elucidating disease mechanisms and developing targeted therapies.

Product Specifications and Quality Considerations

Recombinant Human ATP7B Protein (SKU: rp366473) is produced as a recombinant protein for use in biochemical and cell-based research assays. Researchers should confirm the following specifications directly with Aladdin Scientific prior to experimental use, as lot-specific certificates of analysis provide definitive data:

• Purity: Confirm SDS-PAGE or HPLC-verified purity with the supplier's certificate of analysis.
• Formulation: Verify buffer composition, stabilizers, and protein concentration from the current lot documentation.
• Endotoxin level: For cell-based assays, request endotoxin testing results (LAL assay).
• Biological activity: Confirm any functional activity data (e.g., ATPase activity assay results) available for the specific lot.

Always refer to the product datasheet provided with each shipment or available on the Aladdin Scientific website for the most current and lot-specific information.

Recommended Handling and Storage Protocol

Proper handling and storage of Recombinant Human ATP7B Protein are critical to maintaining protein integrity and experimental reproducibility. Follow these general best practices:

1. Upon receipt: Briefly centrifuge the vial at low speed to collect contents, then place immediately on ice if working within the day, or store according to the datasheet instructions.
2. Storage temperature: For long-term storage, aliquot into single-use volumes and store at −80 °C to avoid repeated freeze-thaw cycles, which can cause protein aggregation and loss of activity.
3. Thawing: Thaw aliquots gently on ice or at 4 °C. Avoid thawing at 37 °C or by vortexing, as this may denature the protein.
4. Carrier protein: If working at low concentrations (<10 μg/mL), consider adding a carrier protein such as 0.1% BSA to reduce non-specific adsorption to tube walls.
5. Avoid repeated freeze-thaw: Prepare single-use aliquots at first thaw; discard unused portions after each experiment rather than refreezing.
6. Working conditions: Perform all manipulations on ice and minimize exposure to room temperature to preserve enzymatic activity.

Specific storage buffer composition and recommended working concentrations should be confirmed from the product datasheet or by contacting Aladdin Scientific technical support.

Key Research Applications

Recombinant Human ATP7B Protein is a valuable reagent for multiple research contexts:

• Wilson Disease Mechanistic Studies: Use as a reference protein to study the impact of disease-associated missense mutations on protein folding, copper binding, and ATPase activity.
• Copper Homeostasis Research: Investigate the molecular mechanisms by which ATP7B traffics between the trans-Golgi network and sub-apical vesicles in hepatocytes in response to copper levels.
• Enzyme Activity Assays: Employ in ATPase activity assays to characterize inhibitors or activators of copper-transporting ATPase function for drug discovery efforts.
• Antibody Validation: Use as a positive control antigen to validate anti-ATP7B antibodies in Western blot, ELISA, or immunoprecipitation experiments.
• Protein-Protein Interaction Studies: Apply in pull-down or co-immunoprecipitation assays to identify novel interaction partners of ATP7B relevant to copper trafficking pathways.
• Structural Studies: Utilize as a substrate for cryo-EM, X-ray crystallography support studies, or hydrogen-deuterium exchange mass spectrometry (HDX-MS) investigations of conformational dynamics.

Troubleshooting Common Experimental Issues

Researchers may encounter the following challenges when working with Recombinant Human ATP7B Protein. The guidance below reflects general best practices for recombinant P-type ATPase proteins:

Issue	Possible Cause	Recommended Action
Low or absent ATPase activity	Protein denaturation from freeze-thaw cycling or improper thawing	Use freshly thawed single-use aliquots; thaw on ice; verify copper and Mg²⁺-ATP concentrations in assay buffer
Protein aggregation / precipitation	Freezing without cryoprotectant, high concentration stock	Add glycerol (5–10%) to storage buffer; dilute protein before use; filter aggregates with 0.22 µm spin filter
No signal on Western blot	Incorrect antibody, insufficient protein loading, or protein running anomalously	Confirm antibody specificity; load 0.1–1 µg recombinant protein as positive control; note that membrane proteins may run anomalously on SDS-PAGE
High background in ELISA	Non-specific binding of carrier protein or impurities	Optimize blocking conditions; use low-endotoxin BSA; titrate protein concentration

If issues persist, contact Aladdin Scientific technical support with your lot number, experimental conditions, and observed results for tailored assistance.

Frequently asked questions

What is the biological function of the ATP7B protein?

ATP7B is a P1B-type copper-transporting ATPase primarily expressed in the liver. It incorporates copper into ceruloplasmin in the trans-Golgi network and exports excess intracellular copper into bile. Mutations in the ATP7B gene cause Wilson disease, an autosomal recessive disorder of copper overload.

How should Recombinant Human ATP7B Protein (rp366473) be stored to maintain activity?

The protein should be aliquoted into single-use volumes upon first thaw and stored at −80 °C for long-term preservation. Avoid repeated freeze-thaw cycles, which degrade protein integrity. Thaw gently on ice or at 4 °C immediately before use, and keep samples on ice during experiments.

What assays is Recombinant Human ATP7B Protein suitable for?

This protein is suitable for ATPase enzyme activity assays, Western blot positive controls, ELISA, immunoprecipitation, protein-protein interaction studies (pull-down assays), and as an antigen for antibody validation. It may also be used in structural biology applications such as cryo-EM support studies.

Why might ATP7B run at an unexpected molecular weight on SDS-PAGE?

As a multi-transmembrane domain protein, ATP7B may exhibit anomalous migration on SDS-PAGE due to incomplete denaturation of hydrophobic transmembrane segments or glycosylation. Always verify identity using a validated anti-ATP7B antibody on Western blot rather than relying solely on apparent molecular weight.

How can I confirm the biological activity of my ATP7B protein lot?

Request the lot-specific certificate of analysis from Aladdin Scientific, which should include any available functional activity data such as ATPase activity measurements. You can also perform an in-house ATPase colorimetric assay measuring inorganic phosphate release in the presence of Mg²⁺-ATP and copper as co-factor. Contact technical support for guidance on assay conditions specific to this product.

References

1. Lutsenko S. Human copper homeostasis: a network of interconnected pathways. Curr Opin Chem Biol. 2010;14(2):211-217. doi:10.1016/j.cbpa.2010.01.003
2. Członkowska A, Litwin T, Dusek P, et al. Wilson disease. Nat Rev Dis Primers. 2018;4(1):21. doi:10.1038/s41572-018-0018-3
3. Tsivkovskii R, MacArthur BC, Lutsenko S. The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner. J Biol Chem. 2001;276(3):2234-2242. doi:10.1074/jbc.M009888200

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