甲醛固定石蜡包埋食管鳞状细胞癌组织蛋白提取方法的比较

焦叶林; 赵云岗; 刘其伟; 阮豪杰; 高社干; 齐义军

doi:10.16098/j.issn.0529-1356.2021.01.023

解剖学报 >

2021 , Vol. 52 >Issue 1: 141 - 145

DOI: https://doi.org/10.16098/j.issn.0529-1356.2021.01.023

技术方法

甲醛固定石蜡包埋食管鳞状细胞癌组织蛋白提取方法的比较

焦叶林 ,
赵云岗 ,
刘其伟 ,
阮豪杰 ,
高社干 ,
齐义军

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1.河南科技大学医学院，临床医学院，第一附属医院，肿瘤医院;河南省肿瘤表观遗传重点实验室，河南洛阳471003; 2.洛阳市第一人民医院病理科，河南洛阳471003; 3.长治市妇幼保健院医学遗传科，山西长治046011; 4. 许昌学院解剖学教研室，河南许昌461000

收稿日期: 2019-07-31

修回日期: 2019-10-30

网络出版日期: 2021-02-06

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Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues

JIAO Ye-Lin ,
ZHAO Yun-Gang ,
LIU Qi-Wei ,
RUAN Hao-Jie ,
GAO She-Gan ,
QI Yi-Jun

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1.He’nan Key Laboratory of Cancer Epigenetics; Cancer Hospital, the First Affiliated Hospital, College of Clinical Medicine, Medical College of He’nan University of Science and Technology, He’nan Luoyang471003, China; 2.Department of Pathology, the First People’s Hospital Luo Yang, He’nan Luoyang471003, China; 3.Department of Medical Genetics, Hospital for Maternity and Children’s Healthcare of Changzhi City, Shanxi Changzhi046011, China; 4. Department of Anatomy, Xuchang University, He’nan Xuchan461000, China

Received date: 2019-07-31

Revised date: 2019-10-30

Online published: 2021-02-06

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摘要

目的探讨从甲醛固定石蜡包埋（FFPE）食管鳞状细胞癌（ESCC）组织中高效率提取蛋白质的方法。方法 6种裂解液和100 ℃、105 ℃两种条件分别提取8例甲醛固定石蜡包埋食管鳞状细胞癌组织蛋白，Bradford法测定蛋白浓度、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、Western blotting和免疫组织化学评价各方法提取组织蛋白的效率和质量，不同蛋白沉淀方法纯化提取蛋白。结果 Laemmli裂解液（4号）100 ℃处理提取癌组织蛋白效率最高，Western blotting检测癌组织中14-3-3σ蛋白表达与免疫组织化学检测结果一致；2倍体积乙腈(含0.1%三氟乙酸)沉淀FFPE提取蛋白能够降低蛋白电泳背景。结论 Laemmli裂解液100 ℃处理可高效提取的甲醛固定石蜡包埋组织蛋白且适用于Western blotting检测蛋白标志物分子，乙腈蛋白沉淀法能够进一步去除残余的交联蛋白质分子。

关键词： 甲醛固定; 石蜡包埋; 蛋白提取; 蛋白纯化; 免疫印迹法

本文引用格式

焦叶林 , 赵云岗 , 刘其伟 , 阮豪杰 , 高社干 , 齐义军 . 甲醛固定石蜡包埋食管鳞状细胞癌组织蛋白提取方法的比较[J]. 解剖学报, 2021 , 52(1) : 141 -145 . DOI: 10.16098/j.issn.0529-1356.2021.01.023

Abstract

Objective To explore protein extraction efficiency from formaldehyde-fixed paraffin embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue samples with different protocols. Methods Six different lysis buffers with 100 ℃ or 105 ℃ treatments were used for protein extraction, followed by evaluation of protein quantity and quality with Bradford, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis, Western blotting and immunohistochemistry (IHC), using 8 FFPE samples of ESCC. Results The optimal method for protein extraction from FFPE ESCC tissue was Laemmli lysis buffer (Buffer 4) treated with 100 ℃ incubation, evidenced by highest amount of protein recovery. Western blotting and IHC method measured consistent 14-3-3σ expression in FFPE ESCC tissue samples. Protein precipitated by two volumes of acetonitrite acetonitrile(ACN) (0.1% trifluoroacetic acid) relative to protein amount reduced background staining on SDS-PAGE gels by commassie staining. Conclusion Laemmli lysis buffer combined with 100 ℃ incubation has the highest protein extraction efficiency from FFPE ESCC tissue samples for Western blotting measurement of protein biomarkers, and ACN protein precipitation can further eliminate residual cross-linked protein by FFPE.

Key words： Formaldehyde-fixed; Paraffin-embeded tissue; Protein extraction; Protein purification; Western blotting

参考文献

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