Protein Protocols Protein Protocols

638 Grassie and Milligan
6. When preparing samples for SDS-PAGE, care must be taken with addition of nucleophilic
reducing agents, such as dithiothreitol (DTT) and 2-mercaptoethanol, since these can cause
the cleavage of thioester linkages. We limit the concentration of DTT in the sample buffer
to 20 mM. This may not be a universal problem, occurring only with certain proteins.
7. Other commercially available solutions for fluorography, or indeed methods based on
salicylate or 2,5 diphenyloxazole (PPO) may be substituted.
8. The confluency of cells required for [ 35 S]metabolic labeling will varying depending on
the speed of growth of the cell line used. For rapidly growing cells, such as fibroblasts,
add radiolabel at 60% confluency; for slow-growing cells, e.g., of neuronal derivation,
add radiolabel when cells are approaching 80–85% confluency.
9. Subsequent immunoprecipitation (see Subheading 3.3.) of [ 35 S] labeled protein can thus
provide controls for immunoprecipitation efficiency and confirm that the lack of immunoprecipitation
of a [ 3 H]palmitate containing polypeptide was not owing to lack of immunoprecipitation
of the relevant polypeptide by the antiserum.
10. It is recommended that heating is not prolonged and does not exceed 80°C.
11. We have found Pansorbin to be a good and cheap alternative to protein A-Sepharose,
especially for preclearing; however, the use of protein A-Sepharose is recommended for
the immunoprecipitation reaction itself, since the use of Pansorbin has been found to give
increased nonspecific background for some antibodies.
12. Preclearing samples before immunoprecipitation removes material that binds nonspecifically
to protein A, thus reducing the background levels in the final sample. This is especially
useful for [ 35 S] radiolabeled material where it may be advisable to preclear for the
maximum 2 h.
13 The length of time of immunoprecipitation should be determined empirically for each
antibody used, in order to minimize the amount of nonspecific material present in the final
sample. For most antibodies with high titer, the shorter the incubation, the less nonspecific
material immunoprecipitated.
14. Time will obviously depend on the levels of expression of the protein and the amount of
[ 3 H]palmitate used. For cell lines that have not been transfected to express high levels of
a particular protein, 30–40 d of exposure is not an unusual period of time.
15. Stability of incorporation of the [ 3 H]radiolabel to treatment with Tris-HCl, but removal by
hydroxylamine under these conditions can be taken to reflect linkage via a thioester bond.
References
1. Milligan, G., Parenti, M., and Magee, A. I. (1995) The dynamic role of palmitoylation in
signal transduction. Trends Biochem. Sci. 20, 181–186.
2. Wedegaertner, P. B. and Bourne, H. R. (1994) Activation and depalmitoylation of G sα.
Cell 77, 1063–1070.
3. Degtyarev, M. Y., Spiegel, A. M., and Jones, T. L. Z. (1993) Increased palmitoylation of
the G s protein α subunit after activation by the β-adrenergic receptor or cholera toxin.
J. Biol. Chem. 268, 23,769–23,772.
4. Mouillac, B., Caron, M., Bonin, H., Dennis, M., and Bouvier, M. (1992) Agonist-modulated
palmitoylation of β 2-adrenergic receptor in Sf9 cells. J. Biol. Chem. 267, 21,733–21,737.
5. Robinson, L. J., Busconi, L., and Michel, T. (1995) Agonist-modulated palmitoylation of
endothelial nitric oxide synthase. J. Biol. Chem. 270, 995–998.
6. Stoffel. R. H., Randall, R. R., Premont, R. T., Lefkowitz, R. J., and Inglese, J. (1994)
Palmitoylation of G protein-coupled receptor kinase, GRK6. Lipid modification diversity
in the GRK family. J. Biol. Chem. 269, 27,791–27,794.