Related Experiment Video
Updated: May 31, 2026
Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction
Published on: August 15, 2013
Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family
Peter C Simons1, Susan M Young, Mark B Carter
1University of New Mexico Center for Molecular Discovery, Department of Pathology, and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA.
Abstract:
The B-cell lymphoma-2 (Bcl-2) family contains six antiapoptotic members, each with a hydrophobic pocket in which Bcl-2 homology region 3 (BH3) helices bind. This binding quenches apoptotic signals from activated BH3 family members. Many tumor cells either have increased expression of one of these six proteins or become overexpressed under treatment. Six fusion proteins made up of glutathione-S-transferase and each of the Bcl-2 members are bound individually to six glutathione bead sets, each set being easily distinguished by its different intensity of red fluorescence. The coated bead sets are washed, combined and incubated with green fluorescent Bim-BH3 peptide and a small molecule in 10-μl wells for 1 h. The green fluorescence signal for each bead set is resolved, and selective inhibitors are expected to reduce the signal for individual bead sets. Each 384-well plate is analyzed in 12 min, measuring 200 of 2,000 beads (∼10%) of each type per well.
