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Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples

Nature Protocols volume 6, pages 943–952 (2011)Cite this article

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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.

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Figure 1: Bead coating and gating.
Figure 2: Finding an inhibitor specific for Bcl-B.
Figure 3: Multiplex binding and competition curves.

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Acknowledgements

This work was supported by NIH grants 1X01 MHO79850-01, CA113318 and U54 MH074425/MH084690, the New Mexico Molecular Libraries Screening Center/University of New Mexico Center for Molecular Discovery (NMMLSC/UNMCMD), and the University of New Mexico Shared Flow Cytometry Resource and Cancer Research and Treatment Center (P30 CA118100). We acknowledge the contributions of R. Halip, C. Bologa and T. Oprea to the analysis of primary and dose-response screens.

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Authors and Affiliations

  1. Department of Pathology, University of New Mexico Center for Molecular Discovery, and Cancer Research and Treatment Center, University of New Mexico Health Sciences Center, Albuquerque, New Mexico, USA

    Peter C Simons, Susan M Young, Mark B Carter, Anna Waller, Bruce S Edwards & Larry A Sklar

  2. Sanford-Burnham Medical Research Institute, La Jolla, California, USA

    Dayong Zhai & John C Reed

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  1. Peter C Simons
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Contributions

P.C.S. devised the method of GSH bead synthesis and assay development and prepared the manuscript; S.M.Y. contributed to assay development and implementation; M.B.C. was involved in instrument programming and maintenance; A.W. developed and analyzed the spreadsheets for screening and dose response; D.Z. was involved in fusion-protein creation and purification; J.C.R. was the leader of the Burnham team investigating Bcl-2 family and helped in conceptualizing the assay; B.S.E. was the leader of NMMLSC/UNMCMD high-throughput screening core and also a co-developer of HyperCyt platform; L.A.S. was the principal investigator of NMMLSC/UNMCMD, a co-developer of the HyperCyt platform, and contributed to target assay conceptualization and manuscript preparation.

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Correspondence to Larry A Sklar.

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Competing interests

B.S.E. and L.A.S. declare competing financial interests as co-inventors of HyperCyt and co-founders of IntelliCyt Corporation.

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Simons, P., Young, S., Carter, M. et al. Simultaneous in vitro molecular screening of protein-peptide interactions by flow cytometry, using six Bcl-2 family proteins as examples. Nat Protoc 6, 943–952 (2011). https://doi.org/10.1038/nprot.2011.339

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