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

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解剖学报 ›› 2021, Vol. 52 ›› Issue (1) : 141-145. DOI: 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^1,2赵云岗³刘其伟⁴ 阮豪杰¹ 高社干¹ 齐义军^1*

作者信息 +

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*

Author information +

文章历史 +

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。

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.

导出引用

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

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

中图分类号： R-735.1

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