3种细胞裂解液提取总蛋白在Western blot中的效果分析

doi:10.3969/j.issn.10073205.2019.03.004

河北医科大学学报

3种细胞裂解液提取总蛋白在Western blot中的效果分析

1.河北医科大学临床学院实验中心，河北石家庄 050031;2.河北师范大学附属中学高中部,河北石家庄 050017；
3.河北医科大学基础医学院生物化学与分子生物学教研室，河北石家庄 050017；
4.河北医科大学第三医院肾内科，河北石家庄 050051

出版日期:2019-03-25 发布日期:2019-03-20
作者简介:庞雅湘（1979-），女，湖南南县人，河北医科大学临床学院实验师，医学硕士，从事实验中心管理研究。
基金资助:
国家自然科学基金项目（81600576）

Analysis of three cell lysates on total protein extraction in Western blot#br#

1.Department of Experimental Center， the School of Clinical, Hebei Medical University, Shijiazhuang
050031, China; 2.Department of High School, Attached Middle School of Hebei Normal University,
Shijiazhuang 050017, China; 3.Department of Biochemistry and Molecular Biology，the School
of Basic Medicine， Hebei Medical University, Shijiazhuang 050017, China; 4.Department of
Nephrology, the Third Hospital of Hebei Medical University, Shijiazhuang 050051, China

Online:2019-03-25 Published:2019-03-20

摘要/Abstract

摘要： ［摘要］〓
〖HTH〗目的〖HTSS〗〖KG*2〗寻找一种适用于培养血管平滑肌细胞总蛋白提取的细胞裂解液配方，以更好地应用于Western blot分析。
〖HTH〗方法〖HTSS〗〖KG*2〗采用3种常用的细胞裂解液（Ⅰ液，Ⅱ液和Ⅲ液），提取总蛋白后Bradford法测定蛋白含量，SDS聚丙烯酰胺凝胶电泳分离提取的蛋白，Western blot检测PDGFRβ、Akt、GAPDH和SM22α等不同分子量的蛋白表达情况。
〖HTH〗结果〖HTSS〗〖KG*2〗3种细胞裂解液得到的蛋白含量差异无统计学意义（P＞005）。小分子量蛋白（<50 000），细胞裂解液Ⅱ和细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ（P＜0.05）；中等分子量蛋白（50 000~80 000），细胞裂解液Ⅱ和细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ，细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅱ（P＜005）；高分子量蛋白（>80 000），细胞裂解液Ⅲ得到的光密度值高于细胞裂解液Ⅰ（P＜005）。Western blot结果显示：PDGFRβ，细胞裂解液Ⅱ和细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅰ（P＜0.05）； Akt，细胞裂解液Ⅱ和细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅰ，细胞裂解液Ⅲ所检测到的条带光密度值显著高于细胞裂解液Ⅱ（P＜0.05）； GAPDH，细胞裂解液Ⅱ所检测到的条带光密度值显著高于细胞裂解液Ⅰ和细胞裂解液Ⅲ（P＜0.05）；SM22α，细胞裂解液Ⅱ所检测到的条带光密度值显著高于细胞裂解液Ⅰ和细胞裂解液Ⅲ（P＜005）。
〖HTH〗结论〖HTSS〗〖KG*2〗确定了一种裂解效果更好、能满足对不同分子量蛋白进行Western blot检测的裂解液配方，为以Western blot为主的蛋白检测相关研究提供了一定的参考。

关键词: 肌细胞, 平滑肌, 细胞培养技术, 细胞裂解液

Abstract: ［Abstract］〓Objective〖HTSS〗〓To find a cell lysate suitable for total protein extract from cultured vascular smooth muscle cells for better application in Western blot analysis.
〖HTH〗〖WTHZ〗Methods〖HTSS〗〓Three commonly used cell lysates, solution Ⅰ, solution Ⅱ and solution Ⅲ supplement with protease inhibitors were used to extract total protein from cultured vascular smooth muscle cells. Bradford method was used to determine protein content and SDSpolyacrylamide gel electrophoresis(SDSPAGE) was used to isolate protein. And then, Western blot was used to detect the protein expression of PDGFRβ, Akt, GAPDH and SM22α.
〖HTH〗〖WTHZ〗Results〖HTSS〗〓There was no significant difference in protein concentration among the three cell lysates(P＞005). The optical density values obtaining from cell lysate Ⅱ and Ⅲ were higher than that obtained from cell lysate Ⅰ in small molecular weight proteins(<50 000) detection. The optical density values obtaining from cell lysate Ⅱ and Ⅲ were higher than that obtained from cell lysate Ⅰ, and those values obtaining from cell lysate Ⅲ were higher than that obtained from cell lysate Ⅱ in medium molecular weight proteins(50 000-80 000) detection. The optical density values obtaining from cell lysate Ⅲ were higher than that obtained from cell lysate Ⅰ in high molecular weight protein(>80 000) detection. For Western blot, the band optical density of PDGFRβ detected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysate Ⅰ. The band optical density of Akt detected by cell lysate Ⅱ and Ⅲ was significantly higher than that obtained from cell lysate Ⅰ, and the band optical density of Akt detected from cell lysate Ⅲ was significantly higher than that obtained from cell lysate Ⅱ. The band optical density of GAPDH detected from cell lysate Ⅱ was significantly higher than that obtained from cell lysate Ⅰ and Ⅲ, the band optical density of SM22α detected fromcell lysate Ⅱ was significantly higher than that obtained from cell lysate Ⅰ and Ⅲ(P＜005).
〖HTH〗〖WTHZ〗Conclusion〖HTSS〗〓An optimal cell lysate was selected to detect the different molecular weight protein from cultured vascular smooth muscle cells, which could provide a suitable protocol for the study of protein function based on Western blot assay.

Key words: myocytes, smooth muscle, cell culture techniques, cell lysatessolution

庞雅湘1，聂启昊2，刘晓宁3，王〓帅3，冯〓琦3，牛洪琳4*. 3种细胞裂解液提取总蛋白在Western blot中的效果分析[J]. 河北医科大学学报, doi: 10.3969/j.issn.10073205.2019.03.004.

PANG Yaxiang1, NIE Qihao2, LIU Xiaoning3, WANG Shuai3, FENG Qi3, NIU Honglin4*. Analysis of three cell lysates on total protein extraction in Western blot#br#[J]. Journal of Hebei Medical University, doi: 10.3969/j.issn.10073205.2019.03.004.

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3种细胞裂解液提取总蛋白在Western blot中的效果分析

Analysis of three cell lysates on total protein extraction in Western blot#br#

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