Image_2_SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins.tif (302.74 kB)
Download file

Image_2_SPAAC Pulse-Chase: A Novel Click Chemistry-Based Method to Determine the Half-Life of Cellular Proteins.tif

Download (302.74 kB)
figure
posted on 07.09.2021, 04:28 authored by Trevor M. Morey, Mohammad Ali Esmaeili, Martin L. Duennwald, R. Jane Rylett

Assessing the stability and degradation of proteins is central to the study of cellular biological processes. Here, we describe a novel pulse-chase method to determine the half-life of cellular proteins that overcomes the limitations of other commonly used approaches. This method takes advantage of pulse-labeling of nascent proteins in living cells with the bioorthogonal amino acid L-azidohomoalanine (AHA) that is compatible with click chemistry-based modifications. We validate this method in both mammalian and yeast cells by assessing both over-expressed and endogenous proteins using various fluorescent and chemiluminescent click chemistry-compatible probes. Importantly, while cellular stress responses are induced to a limited extent following live-cell AHA pulse-labeling, we also show that this response does not result in changes in cell viability and growth. Moreover, this method is not compromised by the cytotoxicity evident in other commonly used protein half-life measurement methods and it does not require the use of radioactive amino acids. This new method thus presents a versatile, customizable, and valuable addition to the toolbox available to cell biologists to determine the stability of cellular proteins.

History

References