Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging

Research output: Chapter in Book/Report/Conference proceedingChapter

5 Citations (Scopus)

Abstract

Proteins are the major targets of drug discovery and many of the new drugs are designed to exert their effect by disrupting protein-protein interactions. Validation of the inhibition of molecular interactions is generally done by biochemical methods, however, these are often not feasible when the interaction is not stable enough. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining direct molecular interactions between two molecules in the cell membrane or inside cells in their natural state. Although originally established as a flow cytometric approach, FRET has been adapted for microscopy, allowing for analysis of sub-cellular co-localization at the single cell level. In this chapter, we provide theoretical introduction to the phenomenon of FRET, and a protocol - including labeling techniques, measurement, and evaluation of microscopy images - of the simplest microscopic FRET approach, acceptor photobleaching FRET. This technique is generally usable for studying protein interactions and requires only a standard confocal laser scanning microscope. To demonstrate the value of image based FRET for testing pharmacological disruption of protein-protein interactions, we show how inhibition of the heterodimerization of ErbB2 and ErbB1 by the humanized monoclonal antibody pertuzumab can be validated using this technique.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages165-178
Number of pages14
Volume986
ISBN (Print)9781627033107
DOIs
Publication statusPublished - 2013

Publication series

NameMethods in Molecular Biology
Volume986
ISSN (Print)10643745

Fingerprint

Photobleaching
Fluorescence Resonance Energy Transfer
Pharmacology
Proteins
Microscopy
Antibodies, Monoclonal, Humanized
Drug Discovery
Lasers
Cell Membrane
Pharmaceutical Preparations

Keywords

  • ErbB1
  • ErbB2
  • Förster resonance energy transfer
  • Image cytometry
  • Pertuzumab
  • Protein interactions

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Medicine(all)

Cite this

Roszik, J., Tóth, G., Szöllősi, J., & Vereb, G. (2013). Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging. In Methods in Molecular Biology (Vol. 986, pp. 165-178). (Methods in Molecular Biology; Vol. 986). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-311-4_11

Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging. / Roszik, Janos; Tóth, G.; Szöllősi, J.; Vereb, G.

Methods in Molecular Biology. Vol. 986 Humana Press Inc., 2013. p. 165-178 (Methods in Molecular Biology; Vol. 986).

Research output: Chapter in Book/Report/Conference proceedingChapter

Roszik, J, Tóth, G, Szöllősi, J & Vereb, G 2013, Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging. in Methods in Molecular Biology. vol. 986, Methods in Molecular Biology, vol. 986, Humana Press Inc., pp. 165-178. https://doi.org/10.1007/978-1-62703-311-4_11
Roszik, Janos ; Tóth, G. ; Szöllősi, J. ; Vereb, G. / Validating pharmacological disruption of protein-protein interactions by acceptor photobleaching FRET imaging. Methods in Molecular Biology. Vol. 986 Humana Press Inc., 2013. pp. 165-178 (Methods in Molecular Biology).
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