Protein Protocols Protein Protocols

646 Corsini
Fig. 3. Schematic for the tritium labeling of naturally occurring prenols.
2. The next day, allow the reaction mixture to settle, remove the liquid phase, and filter over
glass wool to remove any remaining MnO 2. Add diethyl ether to the original reaction
vessel, and repeat the process. Combine the two solutions, and concentrate to dryness
under a stream of nitrogen.
3. Chromatograph the material by TLC (7:3 hexanes/diethyl ether, R f 0.35). Two overlapping
bands are present corresponding to the cis- and trans-double bond isomer, the upper
band being the cis-isomer and the lower band being the trans.
4. Remove the lower one-third of the overlapping bands, elute with diethyl ether, dry with
MgSO 4, and concentrate to dryness (yield 20%). If not used immediately, this material
should be stored at –70°C to prevent isomerization of the trans-aldehyde to the equilibrium
mixture (~30% cis).
5. Elute the remaining material, and rechromatograph if desired.
6. Reduce the aldehyde to the alcohol using [ 3 H]NaBH 4. Dissolve 2 mg of the aldehyde in
1 mL absolute ethanol, and add 5 µL 14 N NH 4OH. Dissolve the [ 3 H]NaBH 4 in ethanol at
a concentration of 100 mCi/mL. Add 200 µL of the [ 3 H]NaBH 4 to the aldehyde solution,
shake the solution, and then leave it vented in the hood for at least 4 h.
7. Concentrate the material to dryness in a gentle stream of Ar, take up in diethyl ether, and
place over a column of silica gel overlaid with MgSO 4. Purify the material by TLC
(7:3 hexanes/diethyl ether) to remove unreacted starting material and the small amount of
the cis-isomer that is generated during the reaction. Elute the labeled alcohols from the
silica using diethyl ether. Dry using a stream of Ar.
8. Dissolve the product in ethanol, and bring to a final concentration of 2.1 mM, 2.6 mM,
6.5 mM, or 0.4 mM for labeled 2 cis-GGOH, hexahydrogeranylgeraniol, geraniol, and
tetrahydrofarnesol, respectively.
3.2. Cell Culture Experiments
1. Grow HSF and 3T3 cells in monolayers, and maintain in 100-mm Petri dishes at 37°C in
a humidified atmosphere of 95% air, 5% CO2 in DMEM, pH 7.4, supplemented with 10%
FCS v/v, 1% (v/v) NEAA, penicillin (100 U/mL), and streptomycin (0.1 mg/mL).