Product Overview

Recombinant Human ATP11B Protein (SKU: rp367480) is a member of the P4-ATPase (Type IV ATPase) subfamily, which functions as lipid flippases responsible for translocating phospholipids from the exoplasmic to the cytoplasmic leaflet of cellular membranes. ATP11B plays a role in maintaining phospholipid asymmetry across the plasma membrane and intracellular organelle membranes, a process critical for vesicle trafficking, cell signaling, and apoptosis regulation.

This recombinant protein is produced for research use and is intended for scientists investigating P4-ATPase biology, lipid homeostasis, and membrane-associated enzymatic activities. Researchers in cell biology, biochemistry, and related disciplines will find this reagent suitable for a variety of in vitro experimental applications.

Materials Required

Before beginning, ensure the following materials are available in your laboratory:

• Recombinant Human ATP11B Protein (rp367480, Aladdin Scientific)
• Sterile, nuclease-free deionized water or the reconstitution buffer recommended in the product datasheet
• Microcentrifuge tubes (low-protein-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Vortex mixer or tube rotator
• Refrigerated microcentrifuge
• Dry ice or liquid nitrogen for snap-freezing aliquots
• -80°C freezer for long-term storage
• Appropriate assay buffers compatible with downstream applications (e.g., ATPase activity assay buffer, SDS-PAGE sample buffer)

Note: Always consult the specific product datasheet supplied with your lot for exact buffer compositions, recommended protein concentrations, and validated applications.

Step-by-Step Protocol

Step 1: Preparation
Remove the vial of Recombinant Human ATP11B Protein from storage and allow it to equilibrate on ice for 10–15 minutes. Do not thaw at room temperature to minimize protein degradation.

Step 2: Reconstitution
If the protein is supplied in lyophilized form, briefly centrifuge the vial (1,000 × g, 30 seconds) to collect the powder at the bottom. Add the recommended volume of sterile reconstitution buffer or deionized water as specified in the product datasheet. Gently pipette up and down to dissolve; avoid vigorous vortexing, which may cause denaturation. Allow the solution to stand on ice for 5 minutes and mix gently again.

Step 3: Clarification
Centrifuge the reconstituted solution at 10,000 × g for 5 minutes at 4°C to remove any insoluble aggregates. Transfer the supernatant to a fresh low-protein-binding tube.

Step 4: Protein Quantification
Determine the protein concentration using a validated method such as BCA assay or Bradford assay, using BSA as a standard. Record the measured concentration for downstream experiment normalization.

Step 5: Aliquoting
Divide the protein solution into single-use aliquots to prevent repeated freeze-thaw cycles. Snap-freeze each aliquot in liquid nitrogen or dry ice, then transfer to -80°C for long-term storage.

Step 6: Application Setup
Dilute the protein to the desired working concentration using the appropriate assay buffer immediately before use. Follow the specific conditions required by your downstream assay (e.g., ATPase activity assay, binding assay, or western blot).

Storage, Stability & Quality Control

Storage Conditions: Store Recombinant Human ATP11B Protein at -80°C upon receipt. Short-term storage at -20°C is acceptable if the protein will be used within the period specified in the datasheet. Avoid storing at 4°C for extended periods as this may lead to protein aggregation or activity loss.

Freeze-Thaw Cycles: Minimize freeze-thaw cycles to preserve protein integrity and activity. Pre-aliquoting into single-use volumes is strongly recommended.

Stability: Protein stability depends on buffer composition, pH, and the presence of stabilizing agents such as glycerol or BSA. Refer to the supplied Certificate of Analysis (CoA) for lot-specific stability data.

Quality Control: Each lot of rp367480 is subject to quality control testing. Consult the CoA for purity assessment (typically evaluated by SDS-PAGE or HPLC), endotoxin levels, and biological activity data where applicable.

Handling Precautions: Handle all recombinant proteins on ice. Use appropriate personal protective equipment (PPE). This product is for research use only and is not intended for diagnostic, therapeutic, or clinical applications.

Troubleshooting

The following table addresses common issues encountered when working with Recombinant Human ATP11B Protein:

• Protein precipitation or aggregation: Ensure the protein is thawed slowly on ice. If aggregation persists, centrifuge at 10,000 × g at 4°C and use only the clarified supernatant. Check that the buffer pH and ionic strength are appropriate.
• Low or no ATPase activity: Verify that required cofactors (e.g., Mg²⁺ ions) are present in the assay buffer. Confirm the protein has not been subjected to excess freeze-thaw cycles. Check the assay temperature and incubation time against validated conditions in the literature.
• Unexpected bands on SDS-PAGE: Minor bands may represent post-translational modifications or expression-system-related host proteins. Refer to the CoA for expected purity profile and molecular weight.
• Loss of protein during handling: Use low-protein-binding tubes and tips to minimize adsorption losses, especially at low protein concentrations.
• Inconsistent results between experiments: Ensure aliquots are used consistently. Normalize protein amounts by quantification prior to each experiment.