Dmapp

We used a radioactive-based enzyme assay to characterize the activity of FPPS ^{19 (link), 31 (link)}. The reaction mixture contained 50 mM Tris pH 8, 10 mM MgCl₂, 1 mM TCEP, 50 μM DMAPP (Sigma-Aldrich), 100 μM ¹⁴C-IPP (PerkinElmer) and 140 μg of LmFPPS in a total volume of 500 μl. The reaction was incubated at 37 °C for 3 hours followed with the addition of 1 μl of bacterial alkaline phosphatase (150 U/μl) and overnight incubation at 37 °C to hydrolyze prenyl diphosphates into corresponding alcohols. The alcohols were then extracted with 2 ml of hexane and the volumes were concentrated to 100 μl. The concentrated extracts were analyzed using thin layer chromatography (TLC) by blotting them onto a TLC silica-gel plate (Fisher Scientific) along with the product standards, geraniol and farnesol. Radiolabeled substrates and products were separated by benzene:ethyl acetate (4:1) as the developing solvent. The positions of the standards were revealed with iodine vapor and the radiolabeled solution was blotted onto these spots to further visualize these spots by autoradiography. The plate was then exposed to storage phosphor screen (Molecular Dynamics) overnight and subsequently scanned on a phosphorimager (Amersham).