In vitro Farnesylation

For in vitro labeling, 0.2 n-Ci pH]farnesyl pyrophosphate (Du Pont -New England Nuclear) and 2 ng of the CaaX-tagged protein is added to 20 il of reticulocyte lysate (Promega) as source of farnesyl transferase. The mixture is adjusted to contain 5 mM MgC. To carry out the full modification, 1 jxl of dog pancreatic microsomes (Promega), must also be added. The mixture is then incubated for 30 min at 37 °C. Labeled proteins can be recovered from the reaction mixture by immunoprecipitation with an appropriate antibody adsorbed to protein A-Sepharose, and analyzed by SDS-PAGE and fluorography (Wiedlocha etal., 1992). 

2-20 ng CaaX-tagged protein MgCb to a final concentration of 5 mM 

1 il dog pancreatic microscomes (Promega). (Optional, to carry out the full modification, but not required to obtain labelling) 

---