Protein Protocols Protein Protocols

Radiolabeled Prenyl Alcohols and Anaolgs 651
Fig. 6. Metabolic labeling of prenylated proteins in Swiss 3T3 cells after various treatments.
Experimental conditions as in Fig. 5. Quiescent cells were incubated for 20 h in medium
containing 10% FCS, 40 µM simvastatin, and one of the following: lane 1, 5 µM [ 3 H]-all
trans-GGOH (20 Ci/mmol); lane 2, 5 µM [ 3 H]-cis, trans-GGOH (17.5 Ci/mmol); lane 3, 5 µM
[ 3 H]-6,7,10,11,14,15 hexahydrogeranylgeraniol (17.5 Ci/mmol); lane 4, 5 µM [ 3 H]-tetrahydrofarnesol
(17.5 Ci/mmol); and lane 5, 5 µM [ 3 H]-geraniol (17.5 Ci/mmol). Cell pellets
were delipidated, and equal amounts of cell extract (60 µg cell protein/lane) were analyzed by
SDS-PAGE on 12% gels, and gels were fluorographed. In the experiment shown, 334 × 10 3 , 197
× 10 3 , 247 × 10 3 , 271 × 10 3 , 285 × 10 3 cpm/lane were analyzed for lanes 1–5, respectively.
14. Immerse each stained gel in 200 mL of Amplify, and incubate for 20 min, with rocking.
Without rinsing, dry the treated gel onto heavyweight blotting paper and expose to
preflashed Hyperfilm MP for 7–14 d at –70°C. Exposures of >10 wk may be needed to
visualize low abundance labeled proteins (Fig. 7A,B).
3.3.3. Preparation of the Two-Dimensional Gel Internal Standard,
[ 3H]NEM-Labeled Soybean Trypsin Inhibitor
1. Transfer 100 µL of a freshly prepared solution of 22 mM N-ethylmaleimide (NEM) in
acetonitrile to a 0.5-mL Reacti-Vial, and add 250 µL [ 3H]NEM (1 µCi/ µL in pentane;
56 Ci/mmol). Mix, and let stand for 2 h uncapped in a fume hood to evaporate the pentane.
The final volume will be ~110 µL, and the new specific activity will be 95.4 mCi/mmol.
2. Prepare 1 mg/mL solution of soybean trypsin inhibitor (STI) in 50 mM Tris, pH 8.5, and
reduce the protein by adding DTT (2 mM, final concentration). Incubate overnight at 4°C.
3. Remove excess DTT by applying 300 µL of the reduced STI (in six 50-µL aliquots) to six
SW rotor equibbed, spin columns, and centrifuge for 4 min at 1000 g in a preequilibrated
countertop centrifuge. Prepare spin columns by placing 1 mL bed-volume of Bio Gel
P6-DG (previously rehydrated in 50 mM Tris HCl, pH 7.5) into a 1-mL plastic tuberculinsyringe
equipped with a plug of glass wool, followed by centrifugation for 2 min at 1000 g.