酵母双杂交课件

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Title of the Presentation can go here,*,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Title of the Presentation can go here,*,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Title of the Presentation can go here,*,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Title of the Presentation can go here,*,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Title of the Presentation can go here,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,*,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,酵母双杂交,系统,技术介绍,酵母双杂交系统技术介绍,主要内容,2,酵母双杂交的背景介绍,酵母双杂交的基本原理,Clontech,酵母双杂交系统,常见问题分析,酵母双杂交系统技术介绍,主要内容 2 酵母双杂交的背景介绍酵母双杂交的基本原理Clo,背景介绍,:,关注蛋白质,蛋白质相互作用研究,蛋白组时代,蛋白质功能,生物信息学的发展,3,酵母双杂交系统技术介绍,背景介绍:关注蛋白质蛋白质相互作用研究蛋白组时代蛋白质功能,背景介绍,:,为什么要研究蛋白互作？,发现新的蛋白质和蛋白质的新功能,在细胞体内研究抗原和抗体的相互作用,筛选药物的作用位点,研究药物对蛋白质之间相互作用的影响,建立基因组蛋白连锁图,4,酵母双杂交系统技术介绍,背景介绍:为什么要研究蛋白互作？发现新的蛋白质和蛋白质的新,背景介绍,:Clontech,酵母双系统的功能,发现新的与已知蛋白互作的蛋白,鉴定两个蛋白之间的相互作用,确定蛋白相互作用的区域,19 November 2024,5,酵母双杂交系统技术介绍,背景介绍:Clontech 酵母双系统的功能发现新的与已知,背景介绍,Gene,Function,Gene,Expression,Gene,discovery,19 November 2024,6,酵母双杂交系统技术介绍,背景介绍 GeneGeneGene09 October 20,背景介绍,酵母单杂交,-,蛋白与,DNA,的相互作用,Matchmaker Gold Yeast One-Hybrid Library Screening System,（,630491,）,酵母双杂交,-,蛋白与蛋白的相互作用,Matchmaker Gold Yeast Two-Hybrid System,（,630489,）,Make your own“Mate&Plate”library System(630490),酵母三杂交,三个蛋白之间的相互作用,pBridge Three-Hybrid Vector,（,630404,）,19 November 2024,7,酵母双杂交系统技术介绍,背景介绍 酵母单杂交-蛋白与DNA的相互作用Matchmak,背景介绍,Reporter Gene,酵母单杂交,酵母双杂交,酵母三杂交,19 November 2024,8,酵母双杂交系统技术介绍,Reporter Gene,Reporter Gene,背景介绍 Reporter Gene酵母单杂交酵母双杂交酵母,主要内容,酵母双杂交的背景介绍,酵母双杂交的基本原理,Clontech,酵母双杂交系统,常见问题分析,19 November 2024,9,酵母双杂交系统技术介绍,主要内容酵母双杂交的背景介绍酵母双杂交的基本原理Clonte,基本原理,:,酵母双杂交参考文献,Nature,.1989 Jul 20;340(6230):245-6.,A novel genetic system to detect protein-protein interactions.,Fields S,Song O,.,Department of Microbiology,State University of New York at Stony Brook,Stony Brook 11794.,28000,次引用,19 November 2024,10,酵母双杂交系统技术介绍,基本原理:酵母双杂交参考文献Nature.1989 Ju,基本原理,:GAL4,转录因子的结构,AD,DNA-BD,GAL4,蛋白具有两个功能结构域,BD,结合结构域,AD,激活结构域,19 November 2024,11,酵母双杂交系统技术介绍,基本原理:GAL4转录因子的结构ADDNA-BDGAL4,基本原理,:GAL4,转录因子的分子机理,在,GAL4 based,双杂交系统中,:,正常情况在酵母细胞内,Promoter,gal metabolic gene,GAL UAS,Transcription,AD,BD,19 November 2024,12,酵母双杂交系统技术介绍,Reporter Gene,GAL4 AD,基本原理:GAL4转录因子的分子机理在GAL4 based,基本原理,:,三个启动子，四个报告基因,19 November 2024,获得阳性克隆子,报告基因表达,AbA,抗性筛选,Ade,营养筛选,His,营养筛选,X-a-gal,蓝白筛选,M1,G1,G2,降低假阳性！,19 November 2024,13,酵母双杂交系统技术介绍,基本原理:三个启动子，四个报告基因09 October 2,主要内容,酵母双杂交的背景介绍,酵母双杂交的基本原理,Clontech,酵母双杂交系统,常见问题分析,19 November 2024,14,酵母双杂交系统技术介绍,主要内容酵母双杂交的背景介绍酵母双杂交的基本原理Clonte,酵母双杂交系统,:MM Series,19 November 2024,15,酵母双杂交系统技术介绍,酵母双杂交系统:MM Series09 October 2,Clontech MM,系统的优点,SMART,方法与,同源重组,技术结合构建文库，聪明的酵母自己做克隆。,AbA,抗性筛选的添加，有效抑制背景生长，大大降低假阳性。,文库构建于型酵母菌株,型,Y187,中，方便后续与,a,型菌株,Y2H Gold,进行,mating,直接筛选。,19 November 2024,16,酵母双杂交系统技术介绍,Clontech MM系统的优点SMART方法与同源重组技术,Clontech MM,系统,:,蛋白互作基本流程,验证蛋白互作,自己构建文库：,630490,预制细菌文库：,638802,（传统型）,预制酵母菌文库：,630480,（均一化，通用型）,Clontech,蛋白互作相关试剂盒,文库筛选,文库构建,酵母双杂交系统：,630489,酵母菌转化试剂盒：,630439,酵母培养基（套装）：,630495,插入片段检测：,630497,酵母质粒提取：,630467,体内验证：,630489,630305,体外,Co-IP,验证：,630458,630459,631723,631604,19 November 2024,17,酵母双杂交系统技术介绍,Clontech MM系统:蛋白互作基本流程验证蛋白互作自,文库构建,:Clontech MM,文库构建方法,SMART,合成,cDNA,共转化，涂板,培养，收集菌液,文库分装，冻存,1ml mating,AAAAAAAAA,TTTTTTTT,CCC,AAAAAAAAA,TTTTTTTT,GGG,CCC,AAAAAAAAA,TTTTTTTT,GGG,LD-PCR,CCC,AAAAAAAA,TTTTTTTT,GGG,19 November 2024,18,酵母双杂交系统技术介绍,文库构建:Clontech MM文库构建方法 SMART合,SMART,合成,cDNA,共转化，涂板,培养，收集菌液,文库分装，冻存,1ml mating,聪明的酵母,自己,做克隆,SD/Leu-,Y187,同源重组,SMART,c,DNA,两小时构建文库,19 November 2024,19,酵母双杂交系统技术介绍,文库构建,:Clontech MM,文库构建方法,SMART合成cDNA共转化，涂板 培养，收集菌液 文库分,SMART,合成,cDNA,共转化，涂板,培养，收集菌液,文库分装，冻存,1ml mating,SD/Leu-,19 November 2024,20,酵母双杂交系统技术介绍,文库构建,:Clontech MM,文库构建方法,SMART合成cDNA共转化，涂板 培养，收集菌液 文库分,文库构建,:Clontech MM,自制文库筛选,SMART,