• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Journal of Molecular Biology >Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy.
【24h】

Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy.

机译：使用亲和网格为单粒子电子显微镜制备非组氨酸标签的高分子复合物的策略。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Affinity Grids are electron microscopy (EM) grids with a pre-deposited lipid monolayer containing functionalized nickel-nitrilotriacetic acid lipids. Affinity Grids can be used to prepare His-tagged proteins for single-particle EM from impure solutions or even directly from cell extracts. Here, we introduce the concept of His-tagged adaptor molecules, which eliminate the need for the target protein or complex to be His-tagged. The use of His-tagged protein A as adaptor molecule allows Affinity Grids to be used for the preparation of virtually any protein or complex provided that a specific antibody is available or can be raised against the target protein. The principle is that the Affinity Grid is coated with a specific antibody that is recruited to the grid by His-tagged protein A. The antibody-decorated Affinity Grid can then be used to isolate the target protein directly from a cell extract. We first established this approach by preparing negatively stained specimens of both native ribosomal complexes and ribosomal complexes carrying different purification tags directly from HEK-293T cell extract. We then used the His-tagged protein A/antibody strategy to isolate RNA polymerase II, still bound to native DNA, from HEK-293T cell extract, allowing us to calculate a 25-A-resolution density map by single-particle cryo-EM.
机译：亲和网格是具有预先沉积的脂质单层的电子显微镜（EM）网格，该脂质单层包含功能化的镍-三氮三乙酸脂质。亲和网格可用于从不纯溶液甚至直接从细胞提取物中制备用于单颗粒EM的His标记蛋白。在这里，我们介绍了带有His标记的衔接子分子的概念，该分子无需将目标蛋白或复合物带有His标记的。使用带有His标签的蛋白A作为衔接子分子的前提下，只要可以使用特异性抗体或可以针对靶蛋白产生特异性抗体，Affinity Grids即可用于制备几乎任何蛋白质或复合物。原理是，亲和网格覆盖有特异性抗体，该抗体被His标记的蛋白A募集到网格中。然后，可以将装饰有抗体的亲和网格直接从细胞提取物中分离出目标蛋白。我们首先通过直接从HEK-293T细胞提取物中制备天然核糖体复合物和带有不同纯化标签的核糖体复合物的负染色标本来建立这种方法。然后，我们使用了带有His-tagged蛋白A /抗体的策略，从HEK-293T细胞提取物中分离了仍与天然DNA结合的RNA聚合酶II，从而使我们能够通过单颗粒冷冻EM计算25A分辨率的密度图。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号