Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis

Kameo, Shinsa; Aso, Michiki; Furukawa, Ryo; Matsumae, Renon; Yokono, Makio; Fujita, Tomomichi; Tanaka, Ayumi; Tanaka, Ryouichi; Takabayashi, Atsushi

Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis

Kameo, Shinsa; Aso, Michiki; Furukawa, Ryo et al.

Plant and Cell Physiology, 2021, 62(2), 348-355

Permalink : https://hdl.handle.net/2115/85167

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アクセス権	open access
URI	https://hdl.handle.net/2115/85167
タイトル	Substitution of Deoxycholate with the Amphiphilic Polymer Amphipol A8-35 Improves the Stability of Large Protein Complexes during Native Electrophoresis
著者	Kameo, Shinsa ; Aso, Michiki ; Furukawa, Ryo ; Matsumae, Renon ; Yokono, Makio ; Fujita, Tomomichi ; Tanaka, Ayumi ; Tanaka, Ryouichi ; Takabayashi, Atsushi
著者名 Kameo, Shinsa
著者名 Aso, Michiki
著者名 Furukawa, Ryo
著者名 Matsumae, Renon
著者名 Yokono, Makio
著者名 Fujita, Tomomichi
著者名 Tanaka, Ayumi
e-Rad_Researcher 20311516 著者名 Tanaka, Ryouichi
e-Rad_Researcher 90546417 著者名 Takabayashi, Atsushi
言語	英語
キーワード	Amphipol A8-35
	CN-PAGE
	Photosystem
	Protein complex
発行日	2021-01-05
出版者	Oxford University Press
収録物名	Plant and Cell Physiology
巻(号)	62(2)
ページ	348-355
抄録	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.
資源タイプ	学術雑誌論文
出版タイプ	Accepted Manuscript
権利情報	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.
PISSN	0032-0781
関連情報 (isVersionOf)	出版社版DOI https://doi.org/10.1093/pcp/pcaa165