Journal: Nature protocols

Article Title: Measurement of protein farnesylation and geranylgeranylation in vitro , in cultured cells and in biopsies, and the effects of prenyl transferase inhibitors

doi: 10.1038/nprot.2011.387

Figure Lengend Snippet: Linear flow diagram summarizing the alternative protocols designed to assess protein prenylation and the effects of PTIs on prenylation. The first step of the procedure consists of five different options to prepare biological samples for analysis of protein prenylation. In the second part, we describe five different methods to either quantify enzymatic activity of FT or GGT-1 (option A) or to assess the prenylation state of individual proteins (options B–E). Dual-colored steps (options 2A–C) indicate that these methods are suitable for cultured cells, PBMCs, normal tissue or tumor biopsies. Red arrows show methods that require handling of radiochemicals. Steps 2A–C are designed to determine either PT activity or the prenylation state of select proteins in response to prior exposure to a PTI. Steps 2D–E are primarily designed to determine whether or not a candidate protein undergoes prenylation in intact cells (2D) or in vitro (2E), but they can also be done in the presence or absence of previous drug treatment.

Article Snippet: • Precision balance (many suppliers) • Parafilm or equivalent laboratory film • Vortexer (many suppliers) • Nitrogen gas source • Vacuum concentrator (e.g., SpeedVac) • Water baths (several suppliers) Steps 1A, 1D, 2A and 2E • Biological safety cabinet (Thermo Electron, Forma Class II A2) Steps 1A, 1D • Incubator (Thermo Forma Series II water jacketed CO 2 incubator, model 3310) Steps 1A, 1D • Cell culture plates or dishes (many suppliers) Steps 1A, 1D • Refrigerated tabletop centrifuge for ≤50-ml tubes (Eppendorf, model 5810R) Steps 1A, 1C, 2B and C • Refrigerated tabletop centrifuge for ≤2.0-ml tubes (Eppendorf, model 5417R) Steps 1B and C, 2B and C • Plastic cuvettes (10 mm × 4 mm × 45 mm; Sarstedt, cat. no. 67.742) Steps 2A–C • PowerGen* Model 125 homogenizer (Fisher Scientific, cat. no. 14-359-251) Step1C only • PCR machine (MJ Research, model PTC-200 Peltier Thermo Cycler) Step 1E only • Heatblock Digtl 2-BLK (VWR, cat. no. 12259-052) • Autoradiography film (MidSci, Classic film BX, 5 × 7 or 8 × 10 in, depending on size of gels, cat. no. B×57 or B×810) Steps 2B–D only • X-ray film processer Mini-Medical (AFP ImageWorks, cat. no. 9992305300) Steps 2B–D only • Kodak BioMax MS film (Carestream Health, 8 × 10 in, cat. no. 829-4985) Steps 2D and E only • Kodak TranScreen LE intensifying screen (Carestream Health, 8 × 10 in, cat. no. 162-2034) Steps 2D and E only • Rotator for incubating immunoprecipitation reactions (Boekel Scientific Instruments, Orbitron Rotator II) Step 2D only Step 1B only • Branson sonifier 450 (VWR, cat. no. 33995-590) • Ultracentrifuge Optima L-90K (Beckman Coulter, cat. no. 365670) • SW 55-Ti rotor (Beckman Coulter, cat. no. 342194) • Centrifuge tubes, Ultra-Clear 13 × 51 mm (Beckman Coulter, cat. no. 344057) Step 2A only • GF/B glass fiber filters (25 mm diameter; Whatman, cat. no. 1821-025) • Sampling manifold 12-hole (Millipore, cat. no. XX2702550) • Liquid scintillation counting system (Beckman, model LS 6500) Steps 2B–E only • PVDF membrane Immobilon-P (Millipore, cat. no. IPVH00010) Steps 2B–E • Rocking platform (many suppliers)

Techniques: Activity Assay, Cell Culture, In Vitro