Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Biotechniques. 2006 Jan;40(1):61-6. doi: 10.2144/000112036.


Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.

Publication types

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

MeSH terms

  • Animals
  • Bacterial Proteins / analysis*
  • Bacterial Proteins / chemistry
  • COS Cells
  • Chlorocebus aethiops
  • Fluorescent Dyes / analysis*
  • Fluorescent Dyes / chemistry
  • Genetic Variation
  • Green Fluorescent Proteins / analysis
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / chemistry
  • Microscopy, Fluorescence / methods*
  • Plasmids / physiology
  • Proto-Oncogene Proteins / analysis
  • Proto-Oncogene Proteins / genetics
  • Recombinant Fusion Proteins / analysis
  • Transfection / methods


  • Bacterial Proteins
  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Luminescent Proteins
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • citrine protein, bacteria
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins