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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
| 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)
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Submitter: 芳賀 永
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