Ultima gold f scintillation fluid

Filter binding assay was performed by adapting a previous eIF2 GEF assay (Gomez et al., 2002 (link)). Briefly, purified eIF2B was mixed with 0.1 μl GTP [γ-³²P] (3000 Ci/mmol) (Perkin Elmer) in a final volume of 160 μl (10 mM HEPES pH 7.4, 100 mM KCl, 10 mM MgCl₂) in a glass test tube and incubated at room temperature for 20 min (or the given time). The binding reaction was stopped by applying the reaction to a 0.45 μM cellulose nitrate membrane filter (Whatman WCN 25 mm diameter circles) fitted within a vacuum filter manifold (Millipore) and washed twice with 2.5 ml of ice-cold buffer (10 mM HEPES pH 7.4, 100 mM KCl, 10 mM MgCl₂). Membranes were dried, submerged in 5 ml of Ultima Gold F scintillation fluid (Perkin Elmer) and counted in a Tri-Carb 2100TR liquid scintillation analyzer for 1 minute. Included unlabelled competitor nucleotides were added at indicated concentrations to test off-rate of the bound radiolabelled nucleotide.

Bacteria from overnight cultures grown in BHI medium were adjusted to OD₆₀₀ of 0.05 in fresh BHI and grown at 37°C to an OD₆₀₀ of 0.4. The cultures were harvested at 4°C, washed in ice-cold standard phosphate-buffer saline (pH 7.4) supplemented with 1.6 mM MgSO₄, re-suspended to an OD₆₀₀ of 15 and kept on ice until further analysis. Then, glucose (1% w/v) was added to a 50 μl aliquot, and the cells were allowed to recover for 10 min at 37°C. The transport assay was initiated by the addition of 2 μl of a solution containing the desired concentration of non-labelled L-glutamine and 0.1 μCi of L-[2,3,4-³H] glutamine (60 Ci/mmol) (American Radiolabeled Chemicals, Inc.). For competition assays, the solution contained also the competing amino acid to a working concentration of 900 μM. After incubation at 37°C for the indicated times, 3 ml of ice-cold assay buffer were added and the sample was immediately filtered through a moistened 2.4-cm diameter glass microfiber filter (GF/C; Whatman). The filter was then washed with an additional 3 ml of ice-cold assay buffer, dried and placed in a scintillation vial. 3 ml of Ultima Gold F scintillation fluid (Perkin Elmer) was added, and radioactivity was measured the following day on a β-counter.