利用 NanoBRET 在活细胞中进行广谱激酶谱分析的单示踪剂方案。
Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET.
机构信息
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53719, USA.
Structural Genomics Consortium, Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Straße 15, 60438 Frankfurt am Main, Germany; Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Straße 9, 60438 Frankfurt am Main, Germany.
出版信息
STAR Protoc. 2021 Sep 15;2(4):100822. doi: 10.1016/j.xpro.2021.100822. eCollection 2021 Dec 17.
This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).
本方案用于在活细胞环境中对近 200 种激酶的激酶抑制剂结合情况进行分析。该方案利用单一的激酶示踪剂（NanoBRET(TM) Tracer K10），以四个不同浓度定量运行。与以前使用六种示踪剂组合的方案相比，减少示踪剂的数量可显著改善工作流程。每个 NanoBRET(TM)激酶测定均使用市售质粒构建，并针对 NanoLuc 标记方向、稀释 DNA 和示踪剂浓度进行了优化。如需详细了解本方案的使用和执行情况，请参考 Vasta 等人（2018 年）的文献。
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