Background and Biological Significance of ATP8B1

ATP8B1 (ATPase Phospholipid Transporting 8B1), also known as FIC1, is a member of the P4-ATPase subfamily of P-type ATPases. These enzymes function as phospholipid flippases, catalyzing the ATP-dependent translocation of specific phospholipids — primarily phosphatidylserine and phosphatidylethanolamine — from the outer to the inner leaflet of cellular membranes, thereby maintaining membrane asymmetry.

Mutations in the ATP8B1 gene are associated with progressive familial intrahepatic cholestasis type 1 (PFIC1), also referred to as Byler disease, as well as benign recurrent intrahepatic cholestasis type 1 (BRIC1). These are autosomal recessive hepatic disorders characterized by impaired bile secretion. ATP8B1 is predominantly expressed in the liver, intestine, pancreas, and kidney, and plays a critical role in maintaining hepatocanalicular membrane integrity.

Recombinant Human ATP8B1 Protein enables researchers to investigate the molecular mechanisms underlying phospholipid transport, screen for small-molecule modulators, and study protein–protein interactions relevant to cholestatic liver disease.

Materials and Equipment Required

• Recombinant Human ATP8B1 Protein (SKU: rp366332, Aladdin Scientific)
• Sterile, nuclease-free deionized water or reconstitution buffer as specified on the product datasheet
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Calibrated micropipettes and sterile filtered tips
• Microcentrifuge capable of ≥10,000 × g
• Vortex mixer
• Ice bucket with wet ice or dry ice for cold-chain handling
• -80°C ultralow temperature freezer for long-term storage
• Spectrophotometer or fluorometer (for protein quantification, if required)
• Assay-specific reagents as required by downstream application (e.g., ATPase activity assay kit, phospholipid vesicles)

Note: Always consult the product-specific datasheet supplied with SKU rp366332 for the exact reconstitution buffer composition, recommended protein concentration, and storage conditions, as these may be updated.

Step-by-Step Reconstitution and Handling Protocol

Step 1: Pre-Reconstitution Preparation

1. Remove the vial of Recombinant Human ATP8B1 Protein (rp366332) from -80°C storage and allow it to equilibrate briefly on ice or at 4°C. Do NOT thaw at room temperature rapidly.
2. Briefly centrifuge the vial (≈2,000 × g, 30 seconds) to pellet any lyophilized material that may have adhered to the cap or walls.
3. Prepare all downstream assay components in advance to minimize the time the reconstituted protein remains at ambient temperature.

Step 2: Reconstitution

1. Refer to the product datasheet for the exact recommended reconstitution volume. Typically, reconstitute lyophilized protein by adding sterile deionized water or the specified buffer to achieve the stated stock concentration.
2. Add reconstitution buffer slowly and gently along the inner wall of the vial. Do NOT add directly onto the lyophilized cake.
3. Allow the vial to stand on ice for 5–10 minutes to permit full hydration.
4. Gently swirl or pipette-mix to dissolve completely. Do NOT vortex vigorously, as this may denature the protein.
5. Briefly centrifuge (≈2,000 × g, 30 seconds) to clear any insoluble material.

Step 3: Protein Quantification (Optional but Recommended)

1. Determine the protein concentration using a standard method (e.g., BCA or Bradford assay) using BSA as standard, or measure absorbance at A280 nm if the extinction coefficient is known.
2. Record the concentration and compare with the certificate of analysis (CoA) value supplied with the product.

Step 4: Aliquoting

1. Divide the reconstituted protein into single-use aliquots using low-binding microcentrifuge tubes to prevent adsorption losses.
2. Label each aliquot clearly with protein name, SKU (rp366332), concentration, date of reconstitution, and operator initials.
3. Snap-freeze aliquots in liquid nitrogen or on dry ice, then transfer to -80°C storage immediately.

Step 5: Working Solution Preparation

1. Thaw one aliquot on ice immediately prior to use. Do not refreeze thawed aliquots.
2. Dilute to the desired working concentration using the appropriate assay buffer. Optimize the working concentration empirically for each specific assay system.

Storage, Stability, and Quality Control

Storage Conditions: Store lyophilized Recombinant Human ATP8B1 Protein (rp366332) at -80°C upon receipt. Once reconstituted, store aliquots at -80°C. Short-term storage at 4°C (up to 48 hours) may be acceptable; always refer to the product datasheet for specific stability data.

Freeze-Thaw Cycles: Repeated freeze-thaw cycles significantly compromise protein integrity and enzymatic activity. Single-use aliquoting is strongly recommended to eliminate this risk.

Protein Carrier: Many recombinant proteins are supplied with a carrier protein (e.g., BSA) or glycerol to enhance stability. Verify the formulation in the CoA to ensure compatibility with your downstream assay, particularly if the presence of BSA would interfere with activity measurements.

Quality Control Checks: Before committing the protein to critical assays, perform the following QC steps:

• Confirm protein integrity by SDS-PAGE under reducing conditions, comparing the apparent molecular weight to the expected value for ATP8B1.
• Verify protein identity by Western blot using an anti-ATP8B1 antibody, if available.
• Assess activity using an established ATPase activity assay where applicable.

Expiry and Documentation: Record lot number, expiry date, and CoA values in your laboratory notebook or electronic laboratory information management system (LIMS) for full traceability.

Downstream Applications and Troubleshooting

Recommended Applications: Recombinant Human ATP8B1 Protein (rp366332) is suitable for use in:

• ATPase Activity Assays: Measure inorganic phosphate release to assess phospholipid-stimulated ATPase activity in reconstituted lipid systems.
• Protein–Protein Interaction Studies: ATP8B1 interacts with its beta-subunit CDC50A (TMEM30A); co-immunoprecipitation or pull-down assays can be performed to study this interaction.
• Structural Studies: Suitable for use in biophysical characterization approaches where sufficient protein quantity and purity are confirmed.
• Drug Screening: Use as a target in small-molecule inhibitor or activator screening relevant to cholestatic liver disease research.

Troubleshooting Common Issues: