Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Life Sciences Institute, University of Michigan, Ann Arbor, MI, United States.
Department of Chemistry, University of Michigan, Ann Arbor, MI, United States; Life Sciences Institute, University of Michigan, Ann Arbor, MI, United States.
Methods Enzymol. 2020;640:205-223. doi: 10.1016/bs.mie.2020.04.029. Epub 2020 May 7.
Protein-protein interactions (PPIs) are essential in most biological processes. Even though many methods were designed to detect PPIs, detecting PPIs in a large volume of cells with a temporal resolution remains challenging. Recent development of light gated transcriptional reporters, such as SPARK and iTANGO, enabled detection of PPI in a large population of cells with a temporal resolution on the order of minutes. In this chapter, we discussed in detail the application of SPARK to detect PPIs between the activated β-2 adrenergic receptor (B2AR) and both Gα mimic and β-arrestin2. Because SPARK is a multi-component system, the protein expression level is critical for its optimal performance. We also discussed the detailed protocols for using SPARK with either transfection or lentiviral infection in HEK296T/17 cells.
蛋白质-蛋白质相互作用（PPIs）在大多数生物过程中都是必不可少的。尽管已经设计了许多方法来检测 PPI，但仍难以在具有时间分辨率的大量细胞中检测 PPI。最近开发的光门控转录报告基因，如 SPARK 和 iTANGO，使我们能够以分钟级的时间分辨率在大量细胞中检测 PPI。在本章中，我们详细讨论了 SPARK 在检测激活的β-2 肾上腺素能受体（B2AR）与 Gα模拟物和β-arrestin2 之间的 PPI 中的应用。由于 SPARK 是一个多组分系统，因此其蛋白表达水平对其最佳性能至关重要。我们还讨论了使用 SPARK 在 HEK296T/17 细胞中转染或慢病毒感染的详细方案。
