A method for measuring protein stability. The dynamics of proteins are controlled at several levels, including transcription, translation and degradation. In mammals, the correlation between mRNA levels and protein levels is less clear than in bacteria and single celled eukaryotes, a fact that emphasizes the need for a method for measuring post-transcription control (degradation or translation). Traditional methods for measuring protein half-lives require either radioactive assays that are limited to a few proteins at a time, or the use of protein synthesis inhibitors that cause a severe perturbation to the cells. Furthermore, methods that increase throughput are limited to high-abundant proteins and do not enable real-time monitoring of living cells. The present method “bleach-chase” presents a non-perturbing method for high-throughput measurements of protein degradation and translation rates.
• Simple and robust
• Minimal perturbation to the cell
• Does not stop translation
• Does not require radioactive labeling
• Can be used to monitor the dynamics of degradation
• Can be scaled to measure multiple proteins at high temporal resolution in living cells
Protein dynamics is dictated by production of new proteins and their removal. The latter is the sum of two underlying processes: intra-cellular degradation (e.g. due to proteosome activity) and dilution due to cell growth that effectively reduces the protein amount by 50% every one cell generation. The outlined “bleachchase” technology is aimed to measure protein production and degradation rates. Cells are subjected to a mild pulse of light, which transforms a fraction of their fluorescent proteins into non-fluorescent. The decay of the non-fluorescent protein fraction (the difference between the fluorescence of the bleached and unbleached cells) is then chased in time to reveal the protein half-life: this difference decays in time according to the degradation rate.