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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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Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/63946

Title: An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions
Authors: Ishihara, Seiichiro Browse this author
Mizutani, Takeomi Browse this author
Kawabata, Kazushige Browse this author
Haga, Hisashi Browse this author →KAKEN DB
Keywords: Western blotting
Collagen gel
Serum
Trichloroacetic acid
Collagenase
p65
Issue Date: Jan-2016
Publisher: Springer
Journal Title: Cytotechnology
Volume: 68
Issue: 1
Start Page: 25
End Page: 32
Publisher DOI: 10.1007/s10616-014-9766-4
PMID: 25005915
Abstract: 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
Type: article (author version)
URI: http://hdl.handle.net/2115/63946
Appears in Collections:生命科学院・先端生命科学研究院 (Graduate School of Life Science / Faculty of Advanced Life Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 芳賀 永

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