Product Overview and Background

ATP11C (ATPase Phospholipid Transporting 11C) is a member of the P4-ATPase family, a group of lipid flippases responsible for maintaining the asymmetric distribution of phospholipids across the plasma membrane and intracellular membranes. In humans, ATP11C functions in complex with its beta-subunit CDC50A to catalyze the ATP-dependent translocation of phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer leaflet to the inner leaflet of the lipid bilayer. This asymmetry is critical for cell signaling, apoptosis recognition, and vesicle trafficking.

Loss-of-function mutations or dysregulation of ATP11C have been linked to B-cell lymphopenia, cholestasis, and hemolytic anemia in animal models, making it a biologically significant target for disease mechanism research. Recombinant Human ATP11C Protein (SKU: rp366977) from Aladdin Scientific provides researchers with a reliable, high-purity reagent for in vitro biochemical and functional investigations.

Reconstitution and Storage Protocol

Materials Required: Sterile deionized water or manufacturer-recommended reconstitution buffer, low-binding microcentrifuge tubes, pipettes calibrated for accurate volume delivery.

Reconstitution Steps:

1. Briefly centrifuge the vial at low speed (e.g., 1,000 × g for 30 seconds) to collect lyophilized or liquid material at the bottom of the tube before opening.
2. If the product is supplied as a lyophilized powder, reconstitute by adding the appropriate volume of sterile deionized water or the buffer specified on the Certificate of Analysis (CoA) to achieve the desired working concentration. Gently pipette up and down; avoid vortexing to prevent protein denaturation.
3. Allow the reconstituted protein to equilibrate at room temperature for 5–10 minutes, then mix gently by inversion or slow rotation.
4. Aliquot the reconstituted protein into single-use volumes using low-binding tubes to minimize freeze-thaw losses and protein adsorption.

Storage Conditions:

• Short-term (up to 1 week): Store at 4 °C in working aliquots.
• Long-term: Store at -80 °C. Avoid repeated freeze-thaw cycles, as each cycle can significantly reduce protein activity and integrity.
• Addition of a carrier protein (e.g., 0.1% BSA) to working dilutions may stabilize the protein during storage; verify compatibility with your downstream assay before use.

Note: Always refer to the product-specific Certificate of Analysis (CoA) for confirmed formulation, concentration, and storage recommendations, as these may supersede general guidelines.

Quality Verification and Purity Assessment

Prior to use in functional experiments, verify the quality and integrity of Recombinant Human ATP11C Protein using the following recommended methods:

SDS-PAGE: Run a small aliquot (typically 1–5 µg) under reducing or non-reducing conditions on an appropriate percentage polyacrylamide gel. Stain with Coomassie Brilliant Blue or a silver stain to assess purity. A single predominant band at the expected molecular weight confirms preparation purity. The theoretical molecular weight of the ATP11C protein should be verified against the product CoA.

Western Blot: Confirm protein identity using a validated anti-ATP11C antibody. The detected band should align with the expected molecular weight. This step is particularly recommended when using the protein in novel assay formats or after extended storage.

Endotoxin Testing: For cell-based assays, confirm that endotoxin levels are within acceptable limits (typically <1.0 EU/µg protein) as stated on the CoA, to avoid confounding inflammatory responses in sensitive cellular systems.

Activity Assay (Optional): For functional studies, an ATPase activity assay (e.g., colorimetric phosphate release assay) can be used to confirm enzymatic activity of the recombinant protein in the presence of its beta-subunit (CDC50A) and appropriate phospholipid substrates.

Experimental Applications and Assay Guidelines

Recombinant Human ATP11C Protein is suitable for a range of in vitro research applications. The following guidelines assist in experimental design:

Phospholipid Flippase Activity Assays: ATP11C requires co-expression or co-incubation with its beta-subunit CDC50A for full flippase activity. Reconstitute the protein into liposomes or use detergent-solubilized membrane preparations. Monitor phospholipid translocation using fluorescently labeled phospholipid analogs (e.g., NBD-labeled PS or PE) and measure fluorescence quenching upon dithionite treatment to quantify flipping rates.

Protein–Protein Interaction Studies: Use the recombinant protein in pull-down assays, co-immunoprecipitation, or surface plasmon resonance (SPR) experiments to study interactions between ATP11C and its regulatory partners such as CDC50A/TMEM30A.

Biochemical Characterization: Employ the protein in ATPase activity assays, thermal shift assays (differential scanning fluorimetry, DSF), or circular dichroism (CD) spectroscopy to characterize its biochemical properties and assess the effects of inhibitors or mutations.

Suggested Working Concentrations: Optimal concentrations are assay-dependent. As a starting point, test concentrations in the range of 0.1–10 µg/mL and optimize empirically. Always include appropriate positive and negative controls.

Note: Specific assay conditions, buffer compositions, and co-factor requirements should be optimized based on the experimental system. Refer to peer-reviewed literature for established protocols relevant to P4-ATPase functional studies.

Troubleshooting Common Issues

The following table outlines common issues encountered when working with Recombinant Human ATP11C Protein and suggested corrective actions:

Issue: No or low ATPase activity
Possible Causes: Missing beta-subunit CDC50A; incorrect buffer conditions; protein denaturation from freeze-thaw.
Solutions: Ensure CDC50A is co-present; optimize Mg²⁺ and ATP concentrations; use freshly thawed single-use aliquots.

Issue: Protein precipitation or aggregation
Possible Causes: Freeze-thaw damage; incompatible buffer; concentration too high.
Solutions: Avoid repeated freeze-thaw cycles; clarify by centrifugation (10,000 × g, 10 min, 4 °C) before use; dilute to a lower working concentration; add 0.01–0.1% Tween-20 or BSA to stabilize.

Issue: Multiple bands on SDS-PAGE
Possible Causes: Protein degradation during storage; contamination.
Solutions: Check storage conditions and protein age; ensure vials were maintained at -80 °C without temperature excursions; contact Aladdin Scientific technical support if degradation is observed upon first use.

Issue: High background in functional assay
Possible Causes: Endotoxin contamination; non-specific protein interactions; suboptimal blocking.
Solutions: Confirm endotoxin levels on CoA; optimize blocking conditions; use low-binding plates or tubes.

For persistent issues, contact Aladdin Scientific technical support with the product lot number and CoA for targeted assistance.