Protein Protocols Protein Protocols

664 Andres et al.
Fig. 6. Specific metabolic labeling of RDJ2-transfected HEK cells. (A) Monolayers of human
embryonic kidney HEK cells transfected with pRDJ2 (an expression plasmid that contained the
RDJ2 cDNA under the control of a CMV promoter) were radiolabeled with [ 3 H]mevalonate
and the expressed RDJ2 immunoprecipitated from detergent-solubilized cell extracts using a
RDJ2-specific antibody. A portion of the resulting immunoprecipitate as well as a portion of
the cell extract were subjected to SDS-PAGE. The gel was treated with Amplify, dried, and
exposed to film for 14 d. (B) Immunoprecipitated RDJ2 after metabolic labeling of transfected
HEK cells with [ 3 H]F-OH (lane 1) or [ 3 H]GG-OH (lane 2) was subjected to SDS-PAGE
analysis and fluorography. The remaining immunoprecipitated protein fractions isolated from
HEK cells after metabolic labeling with [ 3 H]F-OH (lane 3) or [ 3 H]GG-OH (lane 4) were
immunoblotted using anti-RDJ2 IgG and subjected to chemiluminescence detection.
3. Methods
3.1. Procedure for the Selective Metabolic Labeling of Farnesylated
and Geranylgeranylated Proteins
Procedures are described for the metabolic labeling of mammalian cells grown to
near-confluence in Falcon 3001 tissue culture dishes. These protocols can be scaled up
or down as appropriate. The incorporation of [ 3 H]F-OH or [ 3 H]GG-OH into protein
was linear with respect to time and the concentration of [ 3 H]isoprenol in tissue culture
dishes ranging from 10 to 35 mm in diameter under the conditions described here
(13,14) (see Note 3).
1. For metabolic labeling experiments with either [ 3 H]F-OH or [ 3 H]GG-OH, a disposable
conical glass centrifuge tube (screw capped) and Teflon-lined cap are flame sterilized.
The labeled isoprenol dissolved in ethanol is added to the tube and the ethanol is evaporated
under a sterile stream of air.
2. An appropriate volume of sterile SS is added to yield a final concentration of 0.5–1 mCi/mL.
The labeled isoprenols are dispersed in the SS by sonication in a Branson bath sonicator
for 10 min. After sonication an aliquot is taken for liquid scintillation counting to verify
that the 3 H-labeled isoprenol has been quantitatively dispersed in SS.