Protocols: Preparation of 14C-Oleate-Albumin
Whole-Cell Cholesterol Esterification
1. 24% (w/v)
In a 50-ml beaker with a stirring bar, add
ml 150 mM NaCl. Slowly
dissolve 6.25 g fatty acid-free BSA (Sigma)
by adding 2 g every 50 min while stirring at
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
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
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
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.
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.