Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis
从小鼠胃肠道中分离上皮细胞进行WB或RNA分析(根据Kulkarni等人1985年发表的试验改进)
发布: 2012年11月20日第2卷第22期 DOI: 10.21769/BioProtoc.292 浏览次数: 22448
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The authors used this protocol in:
May 2012
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Abstract
The gastrointestinal (GI) tract is lined by a single layer of epithelial cells which function in secretion, absorption, and digestion. In addition, most GI tract tumors develop from epithelial cells (carcinomas). This protocol describes isolation of the surface epithelium from the underlying stroma, muscular layer and submucosa in the GI tract. In this protocol, epithelial cell adhesions are weekend by chelating Ca +2 ions followed by mechanical separation of the cells by vortexing. Analysis of protein levels and gene expression patterns in isolated epithelial cells versus whole GI tissue minimizes the potential for confounding contributions from contaminating stromal cells.
Keywords: Intestine tissue (内部组织)
Colon tissue (结肠组织)
Cell isolation (细胞的分离)
Protein (蛋白)
Mouse (小鼠)
Materials and Reagents
- 4% sodium hypochlorite (Sigma-Aldrich, catalog number: 239305 )
- Phosphate buffered saline (PBS) (Mediatech, Cellgro®, catalog number: 21-031 )
- KCl
- NaCl
- KH2PO4
- Na2HPO4
- EDTA
- DTT
- Aprotinin (USB, catalog number: 9087-70-1 )
- Leupeptin (MP, catalog number: 195623 )
- Pepstatin (MP, catalog number: 195368 )
- PMSF (Sigma-Aldrich, catalog number: 329-98-5 )
- Ethanol
- 0.04% sodium hypochlorite (see Recipes)
- 100x protease inhibitor (store at -20 °C) (see Recipes)
- Solution B (see Recipes)
- 1x lysis buffer (see Recipes)
Equipment
- CO2 chamber for mice
- Dissecting scissors (one medium size and one small size for dissecting mice)
- Forceps (2)
- 15-ml conical tubes
- 1.5-ml eppendorf tubes
- 3ml Insulin syringe
- Vortex
- Centrifuge compatible with 15-ml conical
Procedure
文章信息
版权信息
© 2012 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zeineldin, M. and Neufeld, K. (2012). Isolation of Epithelial Cells from Mouse Gastrointestinal Tract for Western Blot or RNA Analysis. Bio-protocol 2(22): e292. DOI: 10.21769/BioProtoc.292.
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分类
分子生物学 > RNA > 转录
生物化学 > 蛋白质 > 免疫检测 > 免疫印迹法(WB )
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