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Ishihara S, Mizutani T, Kawabata K, et al. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions. Cytotechnology. 2014;68(1):25-32doi: 10.1007/s10616-014-9766-4.
Ishihara, S., Mizutani, T., Kawabata, K., & Haga, H. (2016). An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions. Cytotechnology, 68(1), 25-32. https://doi.org/10.1007/s10616-014-9766-4
Ishihara, Seiichiro, et al. "An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions." Cytotechnology vol. 68,1 (2016): 25-32. doi: https://doi.org/10.1007/s10616-014-9766-4
Ishihara S, Mizutani T, Kawabata K, Haga H. An improved method for western blotting when extracting proteins from mammalian cells cultured on a collagen gel under serum-free conditions. Cytotechnology. 2016 Jan;68(1):25-32. doi: 10.1007/s10616-014-9766-4. Epub 2014 Jul 09. PMID: 25005915; PMCID: PMC4698262.
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Cytotechnology. 2016 Jan;68(1):25-32. doi: 10.1007/s10616-014-9766-4. Epub 2014 Jul 09.

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

Cytotechnology

Seiichiro Ishihara, Takeomi Mizutani, Kazushige Kawabata, Hisashi Haga

Affiliations

  1. Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, N10-W8, Kita-ku, Sapporo, 060-0810, Japan.
  2. Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
  3. Transdisciplinary Life Science Course, Faculty of Advanced Life Science, Hokkaido University, N10-W8, Kita-ku, Sapporo, 060-0810, Japan. [email protected].
  4. Research Center for Cooperative Projects, Hokkaido University Graduate School of Medicine, Sapporo, Japan. [email protected].

PMID: 25005915 PMCID: PMC4698262 DOI: 10.1007/s10616-014-9766-4

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-κB 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.

Keywords: Collagen gel; Collagenase; Serum; Trichloroacetic acid; Western blotting; p65

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