Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections
Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections
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DOI：
10.1177/002215549804600314
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发表时间：
1998-03-01
影响因子：
3.2
通讯作者：
Monden, M
中科院分区：
文献类型：
--
作者：
Ikeda, K;Monden, T;Monden, M
我们描述并讨论了从福尔马林固定，石蜡包埋的组织切片中提取蛋白质进行Western印迹分析的方法。从5-mm（2）50-（mu）m厚的组织切片中，将切片在含有2%十二烷基硫酸钠（SDS）的裂解缓冲液中于100 ℃孵育20分钟，然后于60 ℃孵育2小时，可提取大量蛋白质，经SDS-聚丙烯酰胺凝胶电泳（PAGE）鉴定，提取物产生10 - 120 kD的可辨别的蛋白质条带。Western印迹分析成功地检测到膜结合蛋白如E-钙粘蛋白，胞质蛋白如（β）-连环蛋白，以及核蛋白包括增殖细胞核抗原（PCNA）、多克隆型p53、细胞周期蛋白D1、细胞周期蛋白E和细胞周期蛋白依赖性激酶（CDKs）。通过这种技术，我们可以检测细胞周期蛋白D1和CDK 2在小腺瘤中的表达，并与癌组织和正常粘膜进行比较。这里描述的蛋白质提取的简单方法应该可以使用福尔马林固定，石蜡包埋的样品的大规模档案蛋白质印迹分析，其应用可能导致详细的蛋白质表达分析。这项新技术将为分子生物学提供有价值的信息。
We describe and discuss a method of protein extraction for Western Blot analysis from formalin-fixed, paraffin-embedded tissue sections. From 5-mm(2) 50-(mu)meter-thick tissue sections, an abundance of proteins could be extracted by incubating the sections in lysis buffer containing 2% sodium dodecyl sulfate (SDS) at 100C for 20 min followed by incubation at 60C for 2 hr. Extracts yielded discernable protein bands ranging from 10kD to 120kD as identified by SDS-polyacrylamide gel electrophoresis (PAGE). Western blot analysis successfully detected membrane-bound protein such as E-cadherin, cytosolic protein such as (beta)-catenin, and nuclear proteins including proliferating cell nuclear antigen (PCNA), mutant-type p53, cyclin D1, cyclin E, and cyclin-dependent kinases (CDKs). With this technique, we could examine cyclin D1 and CDK2 expression in small adenomas compared with cancer tissues and normal mucosa. The simple method of protein extraction described here should make it possible to use large-scale archives of formalin-fixed, paraffin-embedded samples for Western blot analysis, and its application could lead to detailed analysis of protein expression. This new technique should yield valuable information for molecular biology.