Protocol for quantifying the in vivo rate of protein degradation in mice using a pulse-chase technique

STAR Protoc. 2023 Dec 15;4(4):102574. doi: 10.1016/j.xpro.2023.102574. Epub 2023 Sep 19.

Abstract

The ability to measure the in vivo rate of protein degradation is a major limitation in numerous fields of biology. Here, we present a protocol for quantifying this rate in mice using a pulse-chase technique that utilizes an azide-bearing non-canonical amino acid called azidohomoalanine (AHA). We describe steps for using chow containing AHA to pulse-label the animal's proteome. We then detail the quantification of AHA-labeled proteins in whole-tissue lysates or histological sections using a copper-catalyzed azide-alkyne cycloaddition 'click' reaction. For complete details on the use and execution of this protocol, please refer to Steinert et al. (2023).1.

Keywords: Cell Biology; Metabolism; Molecular Biology; Protein Biochemistry.

MeSH terms

  • Alkynes / chemistry
  • Amino Acids*
  • Animals
  • Azides* / chemistry
  • Mice
  • Proteolysis
  • Proteome

Substances

  • Azides
  • Amino Acids
  • Proteome
  • Alkynes