福建农林大学学报（自然科学版）2018，Vol.47Issue(3)：329-335,7.DOI:10.13323/j.cnki.j.fafu(nat.sci.).2018.03.010
植物叶片染色质免疫共沉淀方法的优化
Optimization of chromatin immunoprecipitation assay in plant leaves
摘要
Abstract
The sonication conditions of chromatin immunoprecipitation (ChIP) for three types of sonicator machine have been ex-plored in this study,and mature leaves of Arabidopsis thaliana were taken as materials. According to the results of ultrasonication, the Covaris M220 Focused-ultrasonicator machine was recommended to obtain 500 bp size DNA fragments with the parameters by 10% duty cycle,75 Watts Intensity Peak Incident power,200 cycles per Burst,7 ℃ bath temperature and 12 min crushing time. Meanwhile,we detected the effect of different doses of H3K9ac antibody on immunoprecipitation efficiency using semi-quantitative PCR and real-time quantitative PCR. It showed that 3 μL H3K9ac antibody was optimal for 0.25 g Arabidopsis leaves. In addition, based on the ultrasonic conditions mentioned above,together with further sonication time optimization, we also obtain suitable size DNA fragments from flag leaves at different senescence stages in rice. Then with an appropriate sample and antibody ratio, DAN samples obtained after immunoprecipitation were sufficient enough for the subsequent ChIP-qPCR and ChIP-sequencing analysis.关键词
叶片/染色质免疫共沉淀/超声破碎条件/抗体用量/免疫沉淀效率Key words
leaf/chromatin immunoprecipitation/sonication conditions/dosage of antibody/immunoprecipitation efficiency分类
生物科学引用本文复制引用
张媛媛,黄冬梅,邓班,李丹静,张玉,缪颖..植物叶片染色质免疫共沉淀方法的优化[J].福建农林大学学报（自然科学版）,2018,47(3):329-335,7.基金项目
福建省科技厅引导性项目(2015N0019) （2015N0019）
国家自然科学基金项目(31470383). （31470383）
