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马艳琴,孙子龙,王彬,$2,陈鹏晓.Western blot检测家兔动脉组织蛋白质表达条件的优化[J].农学学报,2011,(4):52-56..Optimization of Western Blot Condition for Detection of Protein Expression in Rabbit Artery[J].Journal of Agriculture,2011,(4):52-56
Western blot检测家兔动脉组织蛋白质表达条件的优化
Optimization of Western Blot Condition for Detection of Protein Expression in Rabbit Artery
投稿时间:2011-05-23  修订日期:2011-06-21
DOI:
中文关键词: 

冷却电感耦合成像系统


英文关键词: 

Cooled Charged-coupled Device


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基金项目:

氟与铅对动物中枢神经谷氨酸受体通路相关蛋白表达及调控影响的研究

马艳琴  孙子龙  王彬  $2  陈鹏晓
中文摘要:
      

蛋白质免疫印记技术(Western blot)是研究和分析蛋白质的重要手段,由于其步骤繁杂、影响因素众多,常出现背景噪音高、非特异条带多、拖尾等问题,严重影响后续蛋白质定量分析。为建立最佳的检测家兔动脉组织中蛋白质表达的Western blot实验条件,以家兔动脉组织血管细胞粘附分子-1(VCAM-1)为目标蛋白,从电泳条件、抗体浓度、抗体孵育时间、温度及方法、免疫显色方法等方面进行了改良,成功建立了快速、灵敏、特异的western blot体系;并比较了3种常用的酶免疫显色技术:3,3’-二氨基联苯胺(3,3’-diaminobenzidine,DAB)显色、增强化学发光法(enhanced chemilluminescence,ECL)X光片曝光及ECL化学发光冷却电感耦合成像系统(cooled charged-coupled device,CCD)成像的敏感性。结果表明ECL化学发光法比DAB显色法敏感性高出2~10倍,其中CCD系统敏感性高于X光片4倍,且操作简便,是最理想的检测方法。

英文摘要:
      

Western blot is the most important method on studying and analyzing protein. Because it contains many steps, high background, unspecific bands, streaking and so on are the common problems in Western blot, which will influence the subsequent detection and quantitation of protein. To establish the best experiment condition for detection of protein expression in rabbit artery, VCAM-1(vascular cell adhension molecule-1) was detected. A rapid, specific and sensitive procedure has been successfully established, through optimizing several conditions which include SDS-PAGE, antibody dilution, temperature and time for antibody incubation, detection methods and so on. Also, three kinds of detection methods were used which contain DAB, enhanced chemilluminescence (ECL) with exposure captured on X-ray film or with cooled charged-coupled device (CCD) cameras. The sensitivity of three methods was compared. The results indicated that ECL has sensitivity 2-10 times that of DAB, and CCD cameras have 4 times sensitivity of X-ray and a more precise procedure. So, ECL-CCD cameras is a promising detection method of Western blot.

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