Yeast 14C radiotracer uptake (methylammonium)
To test the functionality and the kinetic properties of ammonium transporters, radiotracer experiments can used. 14C-methylammonium is used as a proxy.
1) For uptake measurements, grow yeast cells to logarithmic phase in YNB medium supplemented with 3% glucose and 1 mM L-arginine.
2) Harvest cells at OD600 of 0.6 to 0.8, wash, and resuspend in 40 mM potassium phosphate buffer, pH 7, to a final OD600 of 8.
3) Five minutes before the uptake measurement, supplement cells with 6% glucose and incubate at 30ºC.
4) To start the reaction, add 450 µL of cell suspension to 400 µL of the same buffer containing 200 µM methylammonium and 10 nM 14C-methylammonium (2.11 Gbq/mmol; Amersham).
5) After 0.5, 1, 2, and 4 min, withdraw 200 µL aliquots, dilute in 5 mL ice-cold potassium phosphate buffer containing 200 mM methylammonium, and filter through glass fiber filters (GF/C; Whatman).
6) Wash filters twice with 5 mL of cold deionized water and analyze by liquid scintillation spectrometry.
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Posted 14 Feb, 2007
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