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Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis
| Title: | Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis |
| Authors: | Kameo, Shinsa Browse this author | | Aso, Michiki Browse this author | | Furukawa, Ryo Browse this author | | Matsumae, Renon Browse this author | | Yokono, Makio Browse this author | | Fujita, Tomomichi Browse this author | | Tanaka, Ayumi Browse this author | | Tanaka, Ryouichi Browse this author →KAKEN DB | | Takabayashi, Atsushi Browse this author →KAKEN DB |
| Keywords: | Amphipol A8-35 | | CN-PAGE | | Photosystem | | Protein complex |
| Issue Date: | 5-Jan-2021 |
| Publisher: | Oxford University Press |
| Journal Title: | Plant and Cell Physiology |
| Volume: | 62 |
| Issue: | 2 |
| Start Page: | 348 |
| End Page: | 355 |
| Publisher DOI: | 10.1093/pcp/pcaa165 |
| Abstract: | Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to alpha-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII-LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII-LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PS11 during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes. |
| Rights: | This is a pre-copyedited, author-produced version of an article accepted for publication in Plant and cell physiology following peer review. The version of record is available online at: https://academic.oup.com/pcp/article/62/2/348/6064163 , https://doi.org/10.1093/pcp/pcaa165. |
| Type: | article (author version) |
| URI: | http://hdl.handle.net/2115/85167 |
| Appears in Collections: | 低温科学研究所 (Institute of Low Temperature Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)
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Submitter: 高林 厚史
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