Effect Analysis of Different Cell Lysate on the Extraction of Total Protein form Vascular Tissue and Detection of Phosphor-Protein by Western Blot
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摘要：
为寻找一种适合血管组织蛋白提取的裂解液配方以及用于磷酸化蛋白的免疫印迹分析,运用3种不同的细胞裂解液A,B和C,分别对经预处理的等量猪冠状动脉血管样品进行总蛋白的提取,BCA法测蛋白含量,聚丙烯酰胺凝胶电泳(SDS-PAGE)对蛋白进行分离,免疫印迹法检测pERK,p-AKT,p-MLC和p-MYPT1 4种磷酸化蛋白表达以比较3种裂解液的裂解效果.结果表明,3种裂解液均可以满足一般免疫印迹分析的要求,其中C液提取的蛋白浓度最低,B液提取的蛋白含量最高,但不适于低含量的小分子磷酸化蛋白p-MLC的检测,A液用于免疫印迹检测大、中、小3种分子量的磷酸化蛋白效果最佳.即细胞裂解液A(50mmol·L-1 pH 7.4Tris-HCl,150mmol·L-1 NaCl,0.1%SDS,1%NP-40,1mmol·L-1 EDTA,1mmol·L-1 EGTA,临用前加入蛋白酶抑制剂和磷酸酶抑制剂)可作为提取血管组织总蛋白进一步用于Western检测的一种理想配方.
To search a suitable cell lysate for extraction of total proteins from vascular tissue for detection of phosphor- protein by Western blot. Methods： The proteins were extracted from pretreatment vascular tissue by three different cell lysates： A, B and C. Then total proteins were collected and quantified with the method of BCA assay. The proteins were separated by SDS-PAGE, and the phosphor-protein of pERK, pAKT, pMLC, and pMYPT1 were assessed by Western blot. Results： all samples can be used to detect protein level by Western blot. The concentration of proteins extracted by cell lysate C was low- est. Though the concentration of proteins extracted by cell lysate A were lower than by cell lysate B, the effect of cell lysate A was better than that of cell lysate B used in Western blot, especially in detection of pMLC. Conclusion： The cell lysate A（50 mmol· L-1 pH 7.4 Tris-HCl,150 mmol· L-1 NaCI,0.1% SDS, 1% NP-40,1 mmol· L-1 EDTA,1 mmol· L-1 EGTA, protease and phosphatase inhibitors can be added just before use） is more suitable for extracting proteins from vascular tissue.
作者：
任向波 吴秀丽 李芬
机构地区：
betway官方app 生命科学学院 资源微生物与功能分子河南省高校重点实验室培育基地
出处:
《betway官方app 学报：自然科学版》 CAS 北大核心 2015年第5期102-106,共5页
基金:
国家自然科学基金(30600343) 河南省高校科技创新团队支持计划资助(13IRTSTHN009)
关键词:
细胞裂解液 血管 总蛋白提取 免疫印迹
cell lysate vascular tissue protein extraction western blot
分类号:
Q533 [生物学—生物化学]
