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用于高灵敏度蛋白质检测的多聚化表位标签

Multimerized epitope tags for high-sensitivity protein detection.

作者信息

Stowers R Steven

机构信息

Department of Microbiology and Cell Biology, Montana State University, Bozeman, MT 59717, USA.

出版信息

G3 (Bethesda). 2025 Jun 4;15(6). doi: 10.1093/g3journal/jkaf070.

Abstract

A detailed understanding of the function of a gene requires knowledge of the cellular and subcellular distribution of its encoded protein(s). For proteins expressed at low levels, antibodies that recognize single epitopes may not be sufficient for visualizing expression. To enhance the sensitivity of protein detection, tandem repeat multimers of the commonly used epitope tags V5, HA, MYC, FLAG, ALFA, and OLLAS were developed that encode up to 80X copies of each tag, an 8-fold increase over currently available options for epitope multimer tagging. As proof-of-principle, conditional alleles of vGlut containing the 40XV5 and 40XMYC epitope tag multimers were validated in vivo in Drosophila. Both epitope-tagged proteins were determined to exhibit synaptic localization in the adult brain and larval neuromuscular junction similar to that of endogenous vGlut. They were also conditionally expressed in subsets of adult brain neurons and observed to exhibit robust, easily detectable expression in presynaptic terminals even in single neurons. These highly multimerized epitope tags will facilitate any type of experiment using antibody detection of proteins that would benefit from enhanced sensitivity.

摘要

要详细了解一个基因的功能,需要知道其编码蛋白在细胞和亚细胞水平的分布情况。对于低水平表达的蛋白质,识别单个表位的抗体可能不足以检测到其表达。为提高蛋白质检测的灵敏度,开发了常用表位标签V5、HA、MYC、FLAG、ALFA和OLLAS的串联重复多聚体,每个标签编码多达80倍的拷贝数,比目前用于表位多聚体标记的现有选项增加了8倍。作为原理验证,在果蝇体内验证了含有40X V5和40X MYC表位标签多聚体的vGlut条件等位基因。两种表位标记的蛋白均被确定在成体脑和幼虫神经肌肉接头处表现出与内源性vGlut相似的突触定位。它们也在成体脑神经元亚群中条件性表达,并且观察到即使在单个神经元中,它们在突触前终末也表现出强大、易于检测的表达。这些高度多聚化的表位标签将有助于任何使用抗体检测蛋白质的实验类型,这类实验将受益于更高的灵敏度。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/e7de/12134993/ac7fc460db94/jkaf070f1.jpg

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