Transfer and multiplex immunoblotting of a paraffin embedded tissue.
Transfer and multiplex immunoblotting of a paraffin embedded tissue.
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石蜡包埋组织的转移和多重免疫印迹。
DOI：
10.1002/pmic.200401343
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发表时间：
2006
期刊：
影响因子：
3.4
通讯作者：
Hewitt,StephenM
中科院分区：
文献类型：
--
作者：
Chung,Joon-Yong;Braunschweig,Till;Baibakov,Galina;Galperin,Mike;Ramesh,Arun;Skacel,Marek;Gannot,Gallya;Knezevic,Vladimir;Hewitt,StephenM
随着我们从基因组学过渡到功能蛋白质组的挑战，探索蛋白质在组织内表达的新工具是必不可少的。我们已经开发出一种方法，将蛋白质从福尔马林固定的石蜡包埋组织切片转移到一堆膜上，然后用抗体探测这些膜，以检测单个表位。该方法将传统的组织切片转化为表达谱分析的多路平台。一个组织切片可以转移到多达十个膜上，每个膜用不同的抗体探测，用荧光二次抗体检测，并由微阵列扫描仪进行定量。总蛋白可以在每个膜上测定，因此每个抗体都有自己的归一化。这种方法适用于磷酸特异性抗体，以及不容易与石蜡包埋组织很好地工作的抗体。这项新技术使档案石蜡包埋组织能够以快速和可量化的方式进行分子图谱分析，并将组织微阵列简化为蛋白质阵列的形式。这种方法是探索福尔马林固定的石蜡包埋组织的大量档案的新工具，也是转化医学的工具。
As we transition from genomics to the challenges of the functional proteome, new tools to explore the expression of proteins within tissue are essential. We have developed a method of transferring proteins from a formalin fixed, paraffin embedded tissues section to a stack of membranes which is then probed with antibodies for detection of individual epitopes. This method converts a traditional tissue section into a multiplex platform for expression profiling. A single tissue section can be transferred to up to ten membranes, each of which is probed with different antibodies, and detected with fluorescent secondary antibodies, and quantified by a microarray scanner. Total protein can be determined on each membrane, hence each antibody has its own normalization. This method works with phospho‐specific antibodies as well as antibodies that do not readily work well with paraffin embedded tissue. This novel technique enables archival paraffin embedded tissue to be molecularly profiled in a rapid and quantifiable manner, and reduces the tissue microarray to a form of protein array. This method is a new tool for exploration of the vast archive of formalin fixed, paraffin embedded tissue, as well as a tool for translational medicine.