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

doi:10.16098/j.issn.0529-1356.2021.01.023

解剖学报 ›› 2021, Vol. 52 ›› Issue (1): 141-145.doi: 10.16098/j.issn.0529-1356.2021.01.023

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^1,2赵云岗³刘其伟⁴ 阮豪杰¹ 高社干¹ 齐义军^1*

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 出版日期:2021-02-06 发布日期:2021-02-06
通讯作者: 齐义军 E-mail:qiqiyijun@163.com

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

JIAO Ye-lin^{1, 2} ZHAO Yun-gang³ LIU Qi-wei⁴ RUAN Hao-jie¹ GAO She-gan¹ QI Yi-jun^1*

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:2019-07-31 Revised:2019-10-30 Online:2021-02-06 Published:2021-02-06
Contact: QI Yi-jun E-mail:qiqiyijun@163.com

摘要/Abstract

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

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

中图分类号:

R-735.1

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.

JIAO Ye-lin ZHAO Yun-gang LIU Qi-wei RUAN Hao-jie GAO She-gan QI Yi-jun . Comparison of different protocols for protein extraction from formalin-fixed paraffin embedded esophageal squamous cell carcinoma tissues[J]. Acta Anatomica Sinica, 2021, 52(1): 141-145.

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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

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

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