Protein Protocols Protein Protocols

666 Andres et al.
10. Residual organic solvent is removed from the delipidated protein pellet by evaporation
under a stream of nitrogen. The delipidated protein fractions are then subjected to Pronase E
digestion for analysis of isoprenyl-cysteine analysis by TLC (see Subheading 3.4.), or
dissolved in 2% SDS, 5 mM 2-mercaptoethanol for SDS-PAGE analysis.
3.3. SDS-PAGE Analysis of Metabolically Labeled Proteins
To examine the molecular weight and number of proteins metabolically labeled by
incubation with [ 3H]F-OH or [ 3H]GG-OH, the delipidated protein fractions can be analyzed
by SDS-PAGE. Because these experiments rely on the detection of low-energy
3H-labeled compounds, two procedures are described for the use of fluorography to
increase the sensitivity of detection. (See Note 5 before proceeding.)
1. The delipidated protein fractions are solubilized in 2% SDS, 5 mM β-mercaptoethanol.
An aliquot is used to determine the amount of labeled precursor incorporated into protein.
2. The radiolabeled polypeptides (20–60 µg of protein) were analyzed by SDS-PAGE using
an appropriate percentage polyacrylamide resolving gel (4–20%) for the proteins of interest.
3. Following SDS-PAGE, gels can be analyzed using two distinct methods. In the first, the
gel is directly soaked in the fluorographic reagent, Amplify (Amersham), according to the
manufacturer’s protocol, dried, and exposed to X-ray film as described in step 5. In a
second approach, proteins were electrophoretically transferred to nitrocellulose filters and
stained with Ponceau S to determine the efficiency of transfer. The nitrocellulose filters
are destained by brief washing in distilled water and allowed to air-dry. (See Note 5 for a
discussion of the merits of each method before continuing.)
4. The filters were then dipped briefly in the fluorographic reagent, Amplify (Amersham),
placed on a sheet of plastic backing, and dried for 1 h at 50°C. It is important that a thin
and even film of Amplify reagent remain on the filter and that it be placed protein side
up to dry.
5. Fluorograms were produced by exposing preflashed X-ray film to the nitrocellulose filter,
or dried SDS-PAGE gel, for 5–30 d at –80°C.
3.4. Methods for the Identification of Cysteine-Linked Isoprenyl Group
These simple methods are inexpensive, rapid, and allow the identification of the
isoprenyl-cysteine residue(s) from isoprenylated protein(s). Examples of this method
for the identification of isoprenyl-cysteine groups from metabolically labeled cells and
recombinant proteins labeled in vitro are shown in Figs. 4 and 5. As expected, [ 3H]F-Cys and [ 3H]GG-Cys were liberated from RAS(CVLS) and RAS(CVLL), respectively. A
radioactive peak is also seen at the origin in the analysis of the Pronase digest of radiolabeled
RAS(CVLL) (Fig. 5, middle panel). This radiolabeled product(s) is probably
incompletely digested [ 3H]geranylgeranylated peptides. Rab 1A terminates in two
cysteine residues, both of which are isoprenylated (11). Fig. 5 (lower panel) indicates
that Pronase E is incapable of cleaving between the two cysteine residues.
1. To liberate the labeled isoprenyl-cysteine residues for analysis, the delipidated protein
fractions (50–100 µg) are incubated with 2 mg of Pronase E; 50 mM HEPES, pH 7.4; and
2 mM calcium acetate in a total volume of 0.1 mL at 37°C for approx 16 h. The experimental
scheme for this analysis is illustrated in Fig. 3 (see Note 6).
2. Proteolysis is terminated by the addition of 1 mL of n-butanol saturated with H 2O and
mixing vigorously.