Expanding the repertoire of plasmids for wow PCR-mediated epitope tagging in yeast
Expanding the repertoire of plasmids for wow PCR-mediated epitope tagging in yeast
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DOI：
10.1002/yea.1581
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发表时间：
2008-04-01
期刊：
影响因子：
2.6
通讯作者：
Struhl, Kevin
中科院分区：
文献类型：
--
作者：
Moqtaderi, Zarrnk;Struhl, Kevin
酵母蛋白的表位标记提供了一种在免疫印迹和免疫沉淀实验中跟踪感兴趣的蛋白质的便捷方法，而不需要提高和测试特定的抗体。我们已经构建了四个用于酿酒酵母基于PCR的表位标记的模板。这些质粒扩展了Tag-URA3-Tag环境中可用的表位范围，包括FLAG、HSV、V5和VSV-G表位。所使用的克隆策略将很容易适用于构建基本上针对任何所需表位的类似的Tag-URA3-Tag分子。从一个质粒中设计的用于聚合酶链式反应的寡核苷酸可以与任何其他模板分子互换使用，以允许标记不同的表位，而不需要合成新的引物。我们已经用三个表位标签标记了Tfc6，并评估了这些表位用于染色质免疫沉淀(CHIP)的效率。对于所有标记的等位基因，在已知的Tfc6靶基因上很容易检测到芯片占用信号。这些新标签在需要多个标签蛋白质的实验方案中提供了额外的选择。版权所有(C)2008 John Wiley&Sons，Ltd.
Epitope tagging of yeast proteins provides a convenient means of tracking proteins of interest in Western blots and immunoprecipitation experiments without the need to raise and test specific antibodies. We have constructed four plasmids for use as templates in PCR-based epitope tagging in the yeast Saccharomyces cerevisiae. These plasmids expand the range of epitopes available in a tag-URA3-tag context to include the FLAG, HSV, V5 and VSV-G epitopes. The cloning strategy used would be easily applicable to the construction of a similar tag-URA3-tag molecule for essentially any desired epitope. Oligonucleotides designed for PCR from one plasmid may be used interchangeably with any of the other template molecules to allow tagging with different epitopes without the need for new primer synthesis. We have tagged Tfc6 with each of the triple epitope tags and assessed the efficiency of these epitopes for chromatin immunoprecipitation (ChIP). For all the tagged alleles, ChIP occupancy signals are easily detectable at known Tfc6 target genes. These new tags provide additional options in experimental schemes requiring multiple tagged proteins. Copyright (c) 2008 John Wiley & Sons, Ltd.