title: Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo.
abstract: BACKGROUND
  Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity.
  
  
  RESULTS
  We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target.
  
  
  CONCLUSIONS
  Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications.
authors.affiliation: Research Institute of Molecular Pathology (IMP), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria. benetka@imp.univie.ac.at
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Benetka
authors.forename: Manfred
authors.initials: M
authors.lastname: Koranda
authors.forename: Sebastian
authors.initials: S
authors.lastname: Maurer-Stroh
authors.forename: Fritz
authors.initials: F
authors.lastname: Pittner
authors.forename: Frank
authors.initials: F
authors.lastname: Eisenhaber
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D017368:Protein Prenylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D012680:Sensitivity and Specificity
chemical_list: D011506:Proteins; D019204:GTP-Binding Proteins; C488189:Rasd2 protein, mouse; C442475:Rasd2 protein, rat