Targeted Manipulation/Repositioning of Subcellular Structures and Molecules

Methods Mol Biol. 2019:2038:199-208. doi: 10.1007/978-1-4939-9674-2_13.

Abstract

Technical advances in live-cell imaging have made cell biology into a highly dynamic field, allowing the visualization and quantification of complex processes in individual cells and in real time. To follow changes and to specifically manipulate factors potentially involved in processes like DNA replication, transcription or repair, we set up a universal targeting approach, allowing directed manipulation of subcellular structures and molecules therein. This strategy is based on the very strong and specific interaction of GFP and GFP-binding nanobody. We describe in detail how to set up the targeting approach with appropriate controls, as well as how to improve and validate its efficiency and finally provide exemplary applications.

Keywords: GFP-binding nanobody; Green fluorescent protein; Live-cell microscopy; Protein–protein interaction; Targeted manipulation; Targeted repositioning.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • Microscopy, Fluorescence*
  • Molecular Imaging / methods*
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Protein Binding
  • Single-Cell Analysis / methods*
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / metabolism*

Substances

  • Peptide Fragments
  • Single-Domain Antibodies
  • Green Fluorescent Proteins