title: Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples.
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.
authors: University 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.
authors: Peter C
authors: PC
authors: Simons
authors: Susan M
authors: SM
authors: Young
authors: Mark B
authors: MB
authors: Carter
authors: Anna
authors: A
authors: Waller
authors: Dayong
authors: D
authors: Zhai
authors: John C
authors: JC
authors: Reed
authors: Bruce S
authors: BS
authors: Edwards
authors: Larry A
authors: LA
authors: Sklar
mesh_terms: D019281:Dimerization; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D005982:Glutathione Transferase / Q000737:chemistry; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase