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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 60oC 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 60oC.   Add 10 ml of 150 mM NaCl to the dried oleate soap, gas with argon, cover tightly with aluminum foil, and place in the 60oC 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.

 


Copyright © 2012 Ira Tabas, MD, PhD. All Rights Reserved.