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An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions

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タイトル: An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions
著者: Ishihara, Seiichiro 著作を一覧する
Mizutani, Takeomi 著作を一覧する
Kawabata, Kazushige 著作を一覧する
Haga, Hisashi 著作を一覧する
キーワード: Western blotting
Collagen gel
Serum
Trichloroacetic acid
Collagenase
p65
発行日: 2016年 1月
出版者: Springer
誌名: Cytotechnology
巻: 68
号: 1
開始ページ: 25
終了ページ: 32
出版社 DOI: 10.1007/s10616-014-9766-4
抄録: Western blotting is a widely used method for detection and quantification of specific proteins extracted from mammalian cells. In the conventional method of protein extraction, we found that collagen-containing gels interfered with detection of the p65 protein (one of the subunits in the NF-kappa B family of proteins) in human lung adenocarcinoma A549 cells cultured on a collagen gel containing serum. In contrast, the collagen gels did not affect detection of the GAPDH protein. Then, we established an improved method for preparation of protein extracts (using trichloroacetic acid fixation and collagenase treatment) from the cells cultured on the collagen gel. Using the improved method, we were able to detect p65 proteins without loss in A549 cells cultured on a collagen gel under serum-free conditions, but we could not detect the proteins if serum was present in cell culture. Thus, using western blotting and serum-free culture conditions, we succeeded in comparing the p65 expression between the cells grown in a plastic dish and cells grown on a collagen gel.
Rights: The final publication is available at Springer via http://dx.doi.org/10.1007/s10616-014-9766-4
資料タイプ: article (author version)
URI: http://hdl.handle.net/2115/63946
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 芳賀 永

 

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