To test the functionality and the kinetic properties of ammonium transporters, radiotracer experiments can used. 14C-methylammonium is used as a proxy.
Method Article
Yeast 14C radiotracer uptake (methylammonium)
https://doi.org/10.1038/nprot.2007.87
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To test the functionality and the kinetic properties of ammonium transporters, radiotracer experiments can used. 14C-methylammonium is used as a proxy.
For uptake measurements, grow yeast cells to logarithmic phase in YNB medium supplemented with 3% glucose and 1 mM L-arginine.
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.
Five minutes before the uptake measurement, supplement cells with 6% glucose and incubate at 30ºC.
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).
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).
Wash filters twice with 5 mL of cold deionized water and analyze by liquid scintillation spectrometry.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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