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| Untersuchte Arbeit: Seite: 27, Zeilen: 10-18, 21-26 |
Quelle: Schlüter et al 1999 Seite(n): 411-412, Zeilen: 0 |
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| Incorporation of phenylalanine into cells was analysed by exposing cultures to 14C-phenylalanine for 24 hrs and determining the incorporation of radioactivity into the acid-insoluble cell mass. Non-radioactive phenylalanine (0.3mM) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis.
Experiments were terminated by removal of the medium from the cultures. Cells were washed three times with 1 ml of ice-cold PBS. Subsequently, 1 ml of ice-cold 10% (wt/vol) trichloroacetic acid (TCA) was added. Protein precipitation was performed overnight at 4°C. [...] Radioactivity in this acid fraction presented the intracellular precursor pool. The dishes were then washed twice with 1 ml of ice-cold 10% (wt/vol) trichloroacetic acid and a third time with 1 ml of ice-cold PBS. The remaining precipitate in the culture dishes was dissolved in 1 ml of 1N NaOH/0.01% (wt/vol) SDS by incubation at 37°C overnight. |
[Seite 411]
Incorporation of phenylalanine into cells was determined by exposing cultures to L-14C-phenylalanine (0.1 μCi/ml) for 24 h and determination of the incorporation of radioactivity into acid-insoluble cell mass as described before [7]. [Seite 412] [...] Nonradioactive phenylalanine (0.3 mmol/ l) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis. In incorporation studies, experiments were terminated by removal of the supernatant medium from the cultures and washed three times with ice-cold phosphate-buffered saline (PBS; composition in mmol/l: 1.5 KH2PO4 , 137 NaCl, 2.7 KCl, and 1.0 Na2HPO4, pH 7.4). Subsequently, ice-cold 10% (w/v) trichloroacetic acid was added. After storage overnight at 4°C, the acid was removed from the dishes. Radioactivity contained in this acid fraction was taken to present the intracellular precursor pool. The dishes were then washed twice with ice-cold PBS. The remaining precipitate on the culture dishes was dissolved in 1 M NaOH–0.01% (w/v) sodium dodecylsulphate (S.D.S) by an incubation for 2 h at 37°C. |
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