Determining protein half-lives

Methods Mol Biol. 2004;284:67-77. doi: 10.1385/1-59259-816-1:067.


Controlling the stability of cellular proteins is a fundamental way by which cells regulate growth, differentiation, survival, and development. Measuring the turnover rate of a protein is often the first step in assessing whether or not the function of a protein is regulated by proteolysis under specific physiological conditions. Over the years, procedures to determine the half-life of proteins in cultured eukaryotic cells have been well-established. This chapter describes in detail the two most frequently used methods, pulse-chase analysis and cycloheximide blocking, to determine a protein's half-life in yeast and cultured mammalian cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cycloheximide / pharmacology
  • Cysteine Endopeptidases / metabolism
  • Fungal Proteins / metabolism
  • Half-Life
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Isotope Labeling
  • Methods
  • Multienzyme Complexes / metabolism
  • Promoter Regions, Genetic / drug effects
  • Promoter Regions, Genetic / genetics
  • Proteasome Endopeptidase Complex
  • Protein Processing, Post-Translational*
  • Protein Synthesis Inhibitors / pharmacology
  • Proteins / metabolism*
  • Yeasts / metabolism


  • Fungal Proteins
  • Multienzyme Complexes
  • Protein Synthesis Inhibitors
  • Proteins
  • Cycloheximide
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex