title: Flow cytometric analysis of bimolecular fluorescence complementation: a high throughput quantitative method to study protein-protein interaction.
abstract: Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells. BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.
authors: Department of Pharmacology, University of Illinois at Chicago, USA.
authors: Li
authors: L
authors: Wang
authors: Graeme K
authors: GK
authors: Carnegie
mesh_terms: D001665:Binding Sites; D003584:Cytological Techniques / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D061848:Optical Imaging / Q000379:methods; D060066:Protein Interaction Maps; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011506:Proteins
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