Temporal resolution of protein-protein interactions in the live-cell plasma membrane

Julian Weghuber, Stefan Sunzenauer, Birgit Plochberger, Mario Brameshuber, Thomas Haselgrübler, Gerhard J. Schütz

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

Abstract

We have recently devised a method to quantify interactions between a membrane protein ("bait") and a fluorophore-labeled protein ("prey") directly in the live-cell plasma membrane (Schwarzenbacher et al. Nature Methods 5:1053-1060 2008). The idea is to seed cells on surfaces containing micro-patterned antibodies against the exoplasmic domain of the bait, and monitor the co-patterning of the fluorescent prey via fluorescence microscopy. Here, we characterized the time course of bait and prey micropattern formation upon seeding the cells onto the micro-biochip. Patterns were formed immediately after contact of the cells with the surface. Cells were able to migrate over the chip surface without affecting the micropattern contrast, which remained constant over hours. On single cells, bait contrast may be subject to fluctuations, indicating that the bait can be released from and recaptured on the micropatterns. We conclude that interaction studies can be performed at any time-point ranging from 5 min to several hours post seeding. Monitoring interactions with time opens up the possibility for new assays, which are briefly sketched in the discussion section.

Original languageEnglish
Pages (from-to)3339-3347
Number of pages9
JournalAnalytical and Bioanalytical Chemistry
Volume397
Issue number8
DOIs
Publication statusPublished - Aug 2010

Keywords

  • Atomic force microscopy
  • Fluorescence microscopy
  • Lipid rafts
  • Micro-patterned surfaces
  • Plasma membrane
  • Protein-protein interactions
  • Temporal resolution
  • Cell Line
  • Cells/chemistry
  • Humans
  • Membrane Proteins/chemistry
  • Cell Membrane/chemistry
  • Protein Array Analysis
  • Protein Binding
  • Kinetics

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