Lathouwers Thomas, Wagemans Jeroen, Lavigne Rob
Laboratory of Gene Technology, KU Leuven, Leuven, Belgium.
Methods Mol Biol. 2018;1794:29-48. doi: 10.1007/978-1-4939-7871-7_3.
Array-based yeast two-hybrid (Y2H) screening in combination with a pooling strategy permits identification of complete interaction networks. After discussing the general advantages and drawbacks of Y2H, this chapter provides a detailed protocol for an array-based Gal4p Y2H screen using a pooling strategy to improve efficacy of the experimental work. This includes bait transformation, bait autoactivation testing, pooling of the baits and preys, yeast mating and evaluation of the results. Moreover, a one-on-one confirmation protocol and a quantitative α-galactosidase assay protocol to compare interaction strengths are provided.
基于阵列的酵母双杂交（Y2H）筛选结合混合策略可用于识别完整的相互作用网络。在讨论了Y2H的一般优缺点之后，本章提供了一个基于阵列的Gal4p Y2H筛选的详细方案，该方案使用混合策略来提高实验工作的效率。这包括诱饵转化、诱饵自激活测试、诱饵和猎物的混合、酵母交配以及结果评估。此外，还提供了一对一确认方案和用于比较相互作用强度的定量α-半乳糖苷酶测定方案。
