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

详细信息    查看全文

• 作者：Seiichiro Ishihara ; Takeomi Mizutani ; Kazushige Kawabata ; Hisashi Haga
• 关键词：Western blotting ; Collagen gel ; Serum ; Trichloroacetic acid ; Collagenase ; p65
• 刊名：Cytotechnology
• 出版年：2016
• 出版时间：January 2016
• 年：2016
• 卷：68
• 期：1
• 页码：25-32
• 全文大小：1,474 KB
• 参考文献：Bond MD, Van Wart HE (1984) Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography. Biochemistry 23:3077–3085CrossRef
  Cafruny EJ (1957) Studies on tissue fixation: the penetration of trichloracetic acid solutions into rat tissues. J Histochem Cytochem 5:414–419CrossRef
  Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689CrossRef
  Haga H, Irahara C, Kobayashi R, Nakagaki T, Kawabata K (2005) Collective movement of epithelial cells on a collagen gel substrate. Biophys J 88:2250–2256CrossRef
  Ishihara S, Yasuda M, Harada I, Mizutani T, Kawabata K, Haga H (2013) Substrate stiffness regulates temporary NF-kappaB activation via actomyosin contractions. Exp Cell Res 319:2916–2927CrossRef
  Karin M, Cao Y, Greten FR, Li ZW (2002) NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2:301–310CrossRef
  Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRef
  Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, Fong SF, Csiszar K, Giaccia A, Weninger W, Yamauchi M, Gasser DL, Weaver VM (2009) Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139:891–906
  Li Q, Verma IM (2002) NF-kappaB regulation in the immune system. Nat Rev Immunol 2:725–734CrossRef
  Musial A, Tony Eissa N (2001) Inducible nitric-oxide synthase is regulated by the proteasome degradation pathway. J Biol Chem 276:24268–24273CrossRef
  Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, Gefen A, Reinhart-King CA, Margulies SS, Dembo M, Boettiger D, Hammer DA, Weaver VM (2005) Tensional homeostasis and the malignant phenotype. Cancer Cell 8:241–254
  Peters T (1977) Serum albumin: recent progress in the understanding of its structure and biosynthesis. Clin Chem 23:5–12
  Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354CrossRef
  Tsutsumi K, Tsuda M, Yazawa N, Nakamura H, Ishihara S, Haga H, Yasuda M, Yamazaki R, Shirato H, Kawaguchi H, Nishioka T, Ohba Y (2009) Increased motility and invasiveness in tumor cells that survive 10 Gy irradiation. Cell Struct Funct 34:89–96CrossRef
  Wang YK, Wang YH, Wang CZ, Sung JM, Chiu WT, Lin SH, Chang YH, Tang MJ (2003) Rigidity of collagen fibrils controls collagen gel-induced down-regulation of focal adhesion complex proteins mediated by alpha2beta1 integrin. J Biol Chem 278:21886–21892CrossRef
  Wu X, Guo R, Chen P, Wang Q, Cunningham PN (2009) TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism. Am J Physiol Renal Physiol 297:F316–F326CrossRef
  
• 作者单位：Seiichiro Ishihara (1) (2)
  Takeomi Mizutani (1)
  Kazushige Kawabata (1)
  Hisashi Haga (1) (2)
  
  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
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Biomedicine
  Biochemistry
  
• 出版者：Springer Netherlands
• ISSN：1573-0778

文摘

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 Western blotting Collagen gel Serum Trichloroacetic acid Collagenase p65