Puromycin labeling does not allow protein synthesis to be measured in energy-starved cells

Protein synthesis is a fundamental, tightly regulated cellular process, and several methods have been employed to measure the rate of protein synthesis in cells. One of the most well-established methods entails pulsing cells with radiolabeled amino acids, such as [ 35 S]methionine and [ 35 S]cysteine, for a certain amount of time, and then measuring their incorporation into newly synthesized proteins by quantifying radioactivity. The major disadvantages of this method are that it involves radioactive materials, it is not compatible with fluorescent detection, and it does not allow global identification of newly synthesized proteins by mass spectroscopy. An alternative, nonradioactive approach is based on using the synthetic methionine homolog l -azidohomoalanine (AHA), which harbors an azide group that incorporates into newly synthesized proteins. The use of click chemistry allows us to covalently label the incorporated AHA with reagents, such as biotin, for purification or detection. The amount of biotin-labeled newly synthesized proteins is determined by using tagged streptavidin and immunoblotting, fluorescence-activated cell sorting (FACS), or fluorescent microscopy 1 , 2 .