摘要
为建立植物中特异性免疫沉淀微量蛋白体系,以GUS为报告基因,利用Flag、Myc两种蛋白标签对GUS氨基端进行标记,以农杆菌为介导,在烟草叶片中瞬时高效表达GUS基因。利用Western blot检测叶片中标记的GUS蛋白含量,便于后续GUS蛋白的特异性纯化。以anti-Flag M2磁珠和anti-c-Myc琼脂糖为固相载体,特异性识别和结合标记的GUS蛋白,利用磁分离技术和特异性免疫沉淀对目的蛋白进行两次富集,通过Western blot检测对比各个步骤所得目的蛋白的含量判断富集纯化效果。结果表明,以磁珠和琼脂糖为载体的蛋白,经两次特异性免疫沉淀后得到明显富集和纯化,且每一次的富集效果都较好,初步建立了蛋白标签和磁珠法二次特异性免疫沉淀体系,为体外深入研究活性蛋白复合体奠定了一定的技术基础。
In order to establish specific immunoprecipitation of few proteins in plant,GUS was used as a reporter gene which was marked with Flag and Myc tag on its N-terminal,and was transiently expressed in tobacco leaves via Agrobacterium-mediated method. Western blot analysis suggested the tagged GUS protein accumulated on a large scale in infiltrated tobacco leaves,which was helpful for specific purification of GUS protein. With anti-Flag M2 magnetic beads and anti-c-Myc agarose affinity gel as solid phase carrier,GUS protein was significantly enriched and purified after two-step immunoprecipitation. Western blot analysis indicated that enrichment efficiency of each step of our immunoprecipition was high. The results suggested the specific immunoprecipitation system could largely enrich the amount of tagged GUS protein. Moreover,GUS proteins were pure in final elution. This specific immunoprecipitation system with magnetic beads and protein tags would shed new lights on enrichment and purification of protein complexes of interest.
出处
《中国农业科技导报》
CAS
CSCD
北大核心
2016年第3期184-189,共6页
Journal of Agricultural Science and Technology
基金
国家级大学生创新训练项目(201510019077)
国家自然科学基金项目(31471921)
教育部基本科研业务费专项(2016QC037)资助
关键词
免疫沉淀
磁珠
蛋白标签
微量蛋白
immunoprecipitation
magnetic beads
protein tags
few proteins
