Selected Laboratory Protocols: Preparation of ¹⁴C-Oleate-Albumin Solution for Whole-Cell Cholesterol Esterification Assay

1.  24% (w/v) BSA

       In a 50-ml beaker with a stirring bar, add 17.5 ml 150 mM NaCl.  Slowly dissolve 6.25 g fatty acid-free BSA (Sigma) by adding 2 g every 50 min while stirring at room temperature.  Adjust pH to 7.4 with 5 N NaOH.  Finally, adjust to 25 ml with 150 mM NaCl.  Store at –20⁰C.

2.  12.7 mM Na-oleate-albumin

       Weight out 90 mg oleate (Sigma) by pipetting the oil into a microfuge tube that has been placed in a scintillation vial on a scale. Then transfer the oleate to a 50-ml beaker by using multiple 100-µl absolute EtOH wash-transfers.  Add EtOH so that the final volume of EtOH is 2 ml.  

       Add 100 µL 5N NaOH and mix.  Cover the beaker with aluminum foil and poke small holes in the top.  Remove EtOH by evaporation under a gentle stream of N₂ until completely dry—usually takes 1-3 hours.  Carefully disrupt the soapy paste with a spatula to enable evaporation of EtOH that is trapped in the paste.   NB:  the aluminum foil prevents the dried soap from blowing away.

       Prepare a 60^oC water bath by placing a shallow pool of water in a large beaker on a heating plate and adjusting the temperature until the water is a steady 60^oC.   Add 10 ml of 150 mM NaCl to the dried oleate soap, gas with argon, cover tightly with aluminum foil, and place in the 60^oC water bath.  Stir while heating until clear (10-20 min at 60⁰C). Then stir at room temperature to cool slightly. 

       While still warm, rapidly add 13 ml of ice-cold 24% BSA, gas with argon, cover with parafilm, and stir for a few minutes.  Adjust to 25cc with 150mM NaCl, gas with argon, cover, and stir for few minutes.  Finally, store under argon at ‑20⁰C.  Note that the argon is important because oleate is easily oxidized.

       NB:  The procedure calls for 12.7 mM Na-oleate and 1.79 mM albumin (ratio 7.1 :1), which is close to threshold.  Therefore, if the albumin solution is less than 24% (e.g., due to the loss of some BSA powder during the preparation of the solution), precipitation on cooling of albumin-oleate will result.  To be safe, we usually add an extra 0.3 mg FA-free BSA to the solution.  Test clarity by placing in borosilicate tube and holding up to a light.  In a similar vein, if the solution is cloudy on thawing, add 1cc 24% BSA and stir at 60⁰C for 0.5 hour.

3.  ¹⁴C-oleate-albumin

Add 250 µCi ¹⁴C-oleic acid to a 25-ml Erlenmeyer flask and dry under N₂.  Add 4.35 ml of the Na-oleate-albumin solution prepared above. Then add 1.6 cc 12% BSA in 150 mM NaCl pH=7.4, which was prepared by diluting the 24% BSA solution.  Gas with argon, seal with parafilm, and stir gently at room temperature for 4-6 h.  Filter the solution using a 0.45-µm Millipore filter fitted on a syringe.  Store in aliquots under argon at –20⁰C.  Final oleate concentration is 10 mM.