Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method forVisual Detection of Protein-protein Interactions.
| Autor: | Katoh Y; Department of Physiological Chemistry, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan., Nakamura K; Department of Physiological Chemistry, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan., Nakayama K; Department of Physiological Chemistry, Graduate School and Faculty of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan. |
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| Jazyk: | angličtina |
| Zdroj: | Bio-protocol [Bio Protoc] 2018 Jan 05; Vol. 8 (5), pp. e2687. Date of Electronic Publication: 2018 Jan 05 (Print Publication: 2018). |
| DOI: | 10.21769/BioProtoc.2687 |
| Abstrakt: | The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation ( Katoh et al. , 2015 ). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can be visualized by directly observing the beads bearing immunoprecipitates under a fluorescence microscope. This assay can examine a large number of protein combinations at one time, without requiring time-consuming procedures, including SDS-PAGE and immunoblotting. Furthermore, the VIP assay can examine complicated one-to-many and many-to-many protein interactions. Another important point of the VIP assay is the use of nanobodies for immunoprecipitation. A Nanobody is a single-domain antibody derived from Camelidae (camels and relatives). Because of its small size, high-affinity, high-specificity, and stability, anti-GFP Nanobody expressed in E. coli can be purified on a large scale, and used virtually inexhaustibly for immunoprecipitation experiments. Here we describe protocols for preparation of GST-tagged anti-GFP Nanobody and the VIP assay. (Copyright © 2018 The Authors; exclusive licensee Bio-protocol LLC.) |
| Databáze: | MEDLINE |
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