Protein Protocols Protein Protocols

634 Grassie and Milligan
group to the protooncogene p21 ras is integral both for its membrane association and
transforming activities. Whether there will be similar interest in molecules able to
interfere with protein palmitoylation is more difficult to ascertain as the wide range of
proteins modified by palmitate is likely to limit specificity of such effects. It is true,
however, that compounds able to interfere with protein palmitoylation are available
(8,9). These may prove to be useful experimental reagents in a wide range of studies
designed to explore further the role of protein palmitoylation.
This chapter will describe methodology to determine if a protein expressed in a cell
maintained in tissue culture is in fact modified by the addition of palmitate. Specific
conditions are taken from our own experiences in analysis of the palmitoylation of G
protein α subunits (10–12), but should have universal relevance to studies of protein
palmitoylation.
2. Materials
1. (9,10- 3 H [N])palmitic acid (Dupont/NEN or Amersham International) (see Note 1) and
Trans[ 35 S]-label (ICN Biomedicals, Inc.).
2. Growth medium for fibroblast derived cell lines: DMEM containing 5% newborn calf
serum (NCS), 20 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (Life
Technologies).
3. [ 3 H]palmitate labeling medium: as for growth medium with NCS replaced with 5% dialyzed
NCS (see item 5 below), 5 mM Na pyruvate, and 150 µCi/mL (9,10- 3 H [N]) palmitic
acid (see Note 2). (9,10- 3 H [N])palmitic acid is usually supplied at 1 µCi/mL in ethanol
(see Note 3). Dry under N 2 in a glass tube to remove ethanol, and then redissolve in labeling
medium to give a final concentration of 150 µCi/mL of medium.
4. [ 35 S] methionine/cysteine labeling medium: 1 part growth medium, 3 parts DMEM lacking
methionine and cysteine (Life Technologies) supplemented with 50 µCi/mL
Trans[ 35 S]-label (ICN Biomedicals, Inc.) (see Note 4).
5. Dialyzed NCS: Prepare in dialysis tubing that has been boiled twice in 10 mM EDTA for
10 min (Note 5); 50 mL of NCS are dialyzed against 2 L of Earle’s salts (6.8 g NaCl, 0.1 g
KCl, 0.2 g MgSO 4·7H 2O, 0.14 g NaH 2PO 4, 1.0 g glucose) over a period of 12–36 h with
three changes of buffer. Remove serum from dialysis tubing and filter-sterilize before
storing at –20°C in 2 mL aliquots until required.
6. Phosphate-buffered saline (PBS): 0.2 g KCl, 0.2 g KH 2PO 4, 8 g NaCl, 1.14 g NaHPO 4
(anhydrous) to 1000 mL with H 2O (pH should be in range of 7.0–7.4).
7. 1 and 1.33% (w/v) SDS.
8. TE buffer: 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5.
9. Solubilization buffer: 1% Triton X-100, 10 mM EDTA, 100 mM NaH 2PO 4, 10 mM NaF,
100 µM Na 3VO 4, 50 mM HEPES, pH 7.2.
10. Immunoprecipitation wash buffer: 1% Triton X-100, 0.5% SDS, 100 mM NaCl, 100 mM
NaF, 50 mM NaH 2PO 4, 50 mM HEPES, pH 7.2.
11. Gel solutions for 10% gels:
a. Acrylamide: 30 g acrylamide, 0.8 g bis-acrylamide to 100 mL with H 2O.
b. Buffer 1: 18.17 g Tris, 4 mL 10% SDS (pH 8.8) to 100 mL with H 2O.
c. Buffer 2: 6 g Tris, 4 mL 10% SDS (pH 6.8) to 100 mL with H 2O.
d. 50% (v/v) Glycerol.
e. 10% (w/v) Ammonium persulfate (APS).
f. TEMED.
g. 0.1% (w/v) SDS.