In-Cell Western检测细胞纤连蛋白  

Detection of cellular fibronectin by In-Cell Western

作　　者：李京宝[1] 胡丽芳[1] 张维[1] 骞爱荣[1] 商澎[1] 

机构地区：[1]西北工业大学生命科学院空间生物实验模拟技术国防重点学科实验室,特殊环境生物物理学研究所,陕西西安710072

出　　处：《第四军医大学学报》2009年第19期1861-1863,共3页Journal of the Fourth Military Medical University

基　　金：中国博士后科学基金(20080431249);西北工业大学科技创新基金(2008KJ02039);国家863高技术研究发展计划(2008AA12A218)

摘　　要：目的:利用In-Cell Western技术建立检测细胞纤连白(cFN)的方法.方法:对微孔培养的成骨样细胞进行固定封闭后,使用单克隆抗体对FN进行标记,经红外荧光二抗育后使用Odysssey系统对细胞进行扫描成像和数据分析.在此基础之上优化了固定剂、通透化、封闭剂和一抗浓度.果:免疫印迹法证实了In-Cell Western方法检测的特异性,当MG-63细胞密度低于90%汇合度(细胞数约1.6×104/孔)时,信号与细胞密度呈现较好的线性关系,最低每孔4×103个MG-63细胞的cFN可被检出.结论:成功地建立了一种快速、经济且高通量的cFN检测方法并将其应用于细胞外基质研究.AIM：To establish a method to detect cellular fibronectin（cFN）by In-Cell Western technique.METHODS：Osteoblast like cells（MG-63）cultured in microwells were fixed,blocked,and incubated with anti-FN monoclonal antibody.After being incubated with near-infrared fluorescence labeled secondary antibody,cells were scanned and analyzed by using Odysssey system.The method was further optimized with fixative,permeablization,blocking buffer and concentration of primary antibody.RESULTS：The cFN In-Cell Western method was validated for specificity by traditional western blot under optimized conditions and applied to detect cFN of osteoblast-like cell.The cFN signal of 4×103 cells per well can be detected and the standard curve is linear with MG-63 cell density when cell confluence is lower than 90%（approx.1.6×104 cells per well）.CONCLUSION：A rapid,high-throughput and cost-effective In-Cell Western method for detecting cFN was established and applied to extracellular matrix research successfully.

关 键 词：In—Cell WESTERN 纤连蛋白 细胞外基质 优化 

分 类 号：R445.7[医药卫生—影像医学与核医学]