Protein Protocols Protein Protocols

662 Andres et al.
Fig. 4. Chromatographic analysis of isoprenyl-cysteine residues metabolically labeled by
incubating C6 glioma cells with, [ 3 H]F-OH (upper panel), [ 3 H]GG-OH (middle panel) or
[ 3 H]mevalonate (lower panel). From the traces illustrated, in it can be seen that F-Cys and
GG-Cys are both metabolically labeled when C6 cells are incubated with [ 3 H]mevalonate, but
only F-Cys or GG-Cys are labeled when C6 cells are incubated with [ 3 H]F-OH or [ 3 H]GG-OH,
respectively. Virtually identical results were obtained by chromatographic analysis of the Pronase
E digests of CV-1 proteins metabolically labeled by each isotopic precursor. Radiolabeled
products are also observed at the origin of the TLC which could be incompletely digested
isoprenylated peptides, or in the case of [ 3 H]mevalonate and [ 3 H]GG-OH, possibly mono- or
digeranylgeranylated Cys-Cys or Cys-X-Cys sequences reprinted with permission (ref. 11, Fig. 5).
16. Conical glass tubes.
17. Anisaldehyde spray reagent (26) (see Note 1).
18. Ninhydrin spray reagent (see Note 2).
2.5. Immuniprecipation of Specific Radiolabeled Protein
1. Cell lysis buffer: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1 mM
phenylmethyl+sulfonyl fluoride (PMSF) (add fresh PMSF solution and lysis is carried
out at 4°C.