Protein Protocols Protein Protocols

Protein Palmitoylation 635
Fig. 1. Incorporation of [ 3 H] palmitate into the α subunits of the G proteins G q and G 11. Rat 1
fibroblasts were metabolically labeled with (9,10- 3 H [N])palmitic acid (150 µCi/mL) for the
times indicated (in minutes) as described in Subheading 3.1. The cells were harvested and the
α subunits of the G proteins G q and G 11 were immunoprecipitated as in Subheading 3.3. using
an antipeptide antiserum directed against the C-teminal decapeptide, which is conserved
between these two polypeptides (15). Following SDS-PAGE, the gel was treated as in Subheading
3.5. and exposed to X-ray film for 6 wk.
h. Laemmli sample buffer: 3 g urea, 0.5 g SDS, 0.6 g DTT (see Note 6), 0.5 mL 1 M
Tris-HCl, pH 8.0, to 10 mL with H 2O.
i. Running buffer: 28.9 g glycine, 6 g Tris, 2 g SDS to 2 L with H 2O.
12. Fixing solution: 25% propan-2-ol, 65% H 2O, 10% acetic acid.
13. Amplify (Amersham International plc, UK) (see Note 7).
14. 1 M hydroxylamine adjusted to pH 8.0 with KOH.
3. Methods
3.1. Cell Culture and Metabolic Labeling
1. Seed equal numbers of cells to be analyzed into six-well tissue-culture dishes using
1.5–2 mL growth medium/well (or in 100 mm tissue-culture dishes with 10 mL of
growth medium if cell fractionation is to be carried out). Incubate at 37°C in an atmosphere
of 5% CO 2 until cells are 90% confluent. Remove growth medium and replace
with 1 mL of (9,10- 3 H [N])palmitic acid-labeling medium for 2 h at 37°C (see Fig. 1).
2. Parallel control experiments using Trans[ 35 S]-label (50 µCi/mL) are performed. However,
addition of [ 35 S] methionine/cysteine labeling medium should occur when cell are 60–80%
confluent (see Note 8), and the cells labeled over a period of 18 h (see Note 9).
3.2. Cell Harvesting and Sample Solubilization
1. At the end of the labeling period, remove the labeling medium, and add 200 µL of 1% (w/v)
SDS/well. Scrape the monolayer of cells into the SDS solution, and transfer to a 2 mL
screw-top plastic tube.
2. Heat to 80°C for 20 min (see Note 10) in a heating block to denature proteins. If the
samples have a stringy consistency after this stage, pass through a 20–25 gage needle and
reboil for 10 min.
3. Remove the samples from the heating block, and allow to cool for 2 min at room temperature.
Pulse each tube briefly at high speed in a microfuge to bring the contents to the
bottom of the tube. The samples can either now be frozen at –20°C until required or processed
immediately.