Fluorogenic label to quantify the cytosolic delivery of macromolecules

Mol Biosyst. 2013 Mar;9(3):339-42. doi: 10.1039/c3mb25552b. Epub 2013 Jan 22.

Abstract

The delivery of a macromolecule to the cytosol of human cells is assessed by using a pendant di-O-glycosylated derivative of fluorescein. Its fluorescence is unmasked by Escherichia coliβ-galactosidase installed in the cytosol. Background is diminished by using RNAi to suppress the expression of GLB1, which encodes a lysosomal β-galactosidase. This strategy was used to quantify the cytosolic entry of a highly cationic protein, ribonuclease A.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli Proteins / biosynthesis
  • Fluoresceins / chemistry*
  • Fluoresceins / metabolism
  • Fluorescent Dyes / chemistry*
  • Fluorescent Dyes / metabolism
  • Galactosides / chemistry*
  • Galactosides / metabolism
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Hydrolysis
  • Kinetics
  • Protein Transport
  • RNA, Small Interfering / genetics
  • Ribonuclease, Pancreatic / chemistry
  • Ribonuclease, Pancreatic / metabolism
  • Staining and Labeling / methods
  • beta-Galactosidase / biosynthesis

Substances

  • Escherichia coli Proteins
  • Fluoresceins
  • Fluorescent Dyes
  • Galactosides
  • RNA, Small Interfering
  • fluorescein-digalactoside
  • Ribonuclease, Pancreatic
  • beta-Galactosidase