烟草
免疫沉淀
农杆菌
生物
分子生物学
基因
细胞生物学
遗传学
转基因
作者
Ji Chul Nam,Puneet Bhatt,Sung-Il Kim,Hong-Gu Kang
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 101-110
被引量:1
标识
DOI:10.1007/978-1-0716-3327-4_9
摘要
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.
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