Moqtaderi Zarmik, Struhl Kevin
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Yeast. 2008 Apr;25(4):287-92. doi: 10.1002/yea.1581.
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.
酵母蛋白的表位标签提供了一种在蛋白质印迹法和免疫沉淀实验中追踪目标蛋白的便捷方法，而无需制备和检测特异性抗体。我们构建了四种质粒，用作酿酒酵母中基于PCR的表位标签的模板。这些质粒扩展了在tag-URA3-tag背景下可用的表位范围，包括FLAG、HSV、V5和VSV-G表位。所使用的克隆策略可轻松应用于构建针对基本上任何所需表位的类似tag-URA3-tag分子。从一个质粒设计用于PCR的寡核苷酸可与任何其他模板分子互换使用，从而无需合成新引物即可用不同表位进行标签。我们用每个三重表位标签标记了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靶基因处很容易检测到ChIP占据信号。这些新标签为需要多个标记蛋白的实验方案提供了更多选择。
