Department of Molecular Biosciences, Institute for Cellular & Molecular Biology, The University of Texas at Austin, Austin, TX, USA.
Department of Biology, Texas State University, San Marcos, TX, USA.
Methods Mol Biol. 2023;2690:101-110. doi: 10.1007/978-1-0716-3327-4_9.
The characterization of protein-protein interactions (PPI) often provides functional information about a target protein. Yeast-two-hybrid (Y2H) and luminescence/fluorescence-based detections, therefore, have been widely utilized for assessing PPI. In addition, a co-immunoprecipitation (co-IP) method has also been adopted with transient protein expression in Nicotiana benthamiana (N. benthamiana) infiltrated with Agrobacterium tumefaciens. Herein, we describe a co-IP procedure in which structural maintenance of chromosome 1 (SMC1), identified from a Y2H screening, was verified as an interacting partner for microchidia 1 (MORC1), a protein well known for its function in plant immunity and epigenetics. SMC1 and MORC1 were transiently expressed in N. benthamiana when infiltrated by Agrobacterium with the respective genes. From this approach, we identified a region of SMC1 responsible for interacting with MORC1. The co-IP method, of which outputs are mainly from immunoblot analysis, provided information about target protein expression as well, which is often useful for troubleshooting. Using this feature, we showcased a PPI confirmation from our SMC1-MORC1 study in which a full-length SMC1 protein was not detectable, and, therefore, a subsequent truncated mutant analysis had to be employed for PPI verification.
蛋白质-蛋白质相互作用（PPI）的特征通常提供有关靶蛋白的功能信息。因此，酵母双杂交（Y2H）和基于发光/荧光的检测已被广泛用于评估 PPI。此外，还采用了共免疫沉淀（co-IP）方法，并用瞬时蛋白表达在农杆菌浸润的黄花烟（N. benthamiana）中进行。在此，我们描述了一种 co-IP 程序，其中从 Y2H 筛选中鉴定出的染色体 1 结构维持蛋白 1（SMC1）被验证为 microchidia 1（MORC1）的相互作用伙伴，MORC1 是一种众所周知的植物免疫和表观遗传学功能蛋白。当用携带相应基因的农杆菌浸润时，SMC1 和 MORC1 被瞬时表达在 N. benthamiana 中。通过这种方法，我们确定了 SMC1 与 MORC1 相互作用的区域。co-IP 方法的输出主要来自免疫印迹分析，还提供了有关靶蛋白表达的信息，这对于故障排除通常很有用。使用此功能，我们展示了我们在 SMC1-MORC1 研究中的一个 PPI 确认，其中全长 SMC1 蛋白不可检测，因此必须进行后续的截断突变体分析以进行 PPI 验证。
