Protein degradation kinetics measured by microinjection and live-cell fluorescence microscopy.

ABSTRACT: We have developed a method combining microinjection and automated fluorescence microscopy to continuously assess the degradation rate, subcellular localization and intracellular concentration of protein analytes at the single-cell level. Cells are unperturbed and grown in unaltered environmental conditions and show high viability. The injection of analytes at defined ratios and concentrations allows for a clearly defined starting point of degradation, without the entanglement of biosynthesis/uptake, often encountered in existing methods. The possibility to evaluate, add, or remove post-translational modifications prior to injection represents a powerful tool to assess minute protein degradation rate changes with high precision and allowed us to determine the absolute degradation rates caus

SUBMITTER: Vukovic D 

PROVIDER: S-EPMC11544240 | biostudies-literature | 2024 Nov

REPOSITORIES: biostudies-literature