### Reagents

• Fatty acid–free BSA (Sigma, A6003): 0.64 g

• 5 M NaCl solution (to prepare 150 mM): 60 µL of 5 M NaCl diluted with 1940 µL water

• NaHCO₃: 13.6 mg

• Distilled water (as needed)

• Palmitic acid (Sigma, P5585-10G) or substitute (≈5 mg for a 10 mM final concentration)

• Wako NEFA-HR Assay Kit (for fatty acid measurement)

### Equipment:

• Analytical balance

• Pipettes/micropipettes

• Incubator or water bath (set to 65°C and 37°C)

• Sterile 0.2 µm filter

• Vortex mixer (optional)

• pH meter (if needed)

• 2 mL microcentrifuge tube

### Protocol:

1. Prepare 2 mL of 4.8 mM BSA solution by dissolving 0.64 g BSA in the 150 mM NaCl solution (using 1.5 mL of the prepared 150 mM solution) and adding water to reach a total of 2 mL.

2. Make a saturated NaHCO₃ solution by dissolving 13.6 mg NaHCO₃ in 46 µL distilled water; incubate at 65°C.

3. Add approximately 5 mg palmitic acid (or the equivalent amount for oleic or arachidonic acid) to the NaHCO₃ solution and incubate at 65°C for 2 minutes to form the sodium salt.

4. Transfer 1.6 mL of the BSA solution into the tube containing the fatty acid mixture.

5. Incubate the combined solution at 37°C for 70 minutes.

6. Filter the final mixture through a 0.2 µm filter.

7. Measure the fatty acid concentration with the NEFA-HR assay (expected around 10 mM; roughly a 2:1 fatty acid to BSA molar ratio).