酵母双杂交系统技术介绍.ppt
酵母双杂交系统技术介绍,July 2,2023,宝日医生物技术（北京）有限公司,主要内容,背景介绍:关注蛋白质,背景介绍:为什么要研究蛋白互作？,背景介绍:Clontech 酵母双杂交系统的功能,背景介绍:Clontech分子生物学工具,ClontechMatchmaker,背景介绍:Matchmaker Gold 系统,背景介绍:Matchmaker Gold系统原理,Reporter Gene,Reporter Gene,Reporter Gene,主要内容,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.,63000次引用,基本原理:GAL4转录因子的结构,AD,DNA-BD,BD结合结构域,AD激活结构域,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,Reporter Gene,GAL4 AD,基本原理:三个启动子，四个报告基因,X-a-gal蓝白筛选,M1,G1,G2,降低假阳性！,主要内容,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。,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：630489630305体外Co-IP验证：630458630459631723631604,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,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 cDNA,两小时构建文库,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,SD/Leu-,文库构建:Clontech MM文库构建方法,文库构建:Clontech MM自制文库筛选,SMART合成cDNA,共转化，涂板,培养，收集菌液,文库分装，冻存,1ml mating,Y187，AD 文库,Y2H Gold，BD诱饵培养液,DDO/X/A,混合,QDO/X/A,文库筛选:Clontech MM文库筛选,630489 Matchmaker Gold Yeast Two-Hybrid System,Y187Y2H Gold,三个载体阳性对照，阴性对照。,BD vector：pGBKT7AD vector:pGADT7,630439极大提高转化率可以单独购买,文库筛选:Clontech MM 系统质粒,文库筛选:诱饵载体构建及其自激活检测,Y2H Gold诱饵载体,文库筛选:Mating方法筛选,Y187型,DDO筛选,二倍体,Y2H Golda 型,Mating 接合反应 酵母的有性生殖过程，Clontech利用这个过程，实现了文库筛选。,AD,BD,MixMating,QDO筛选,文库筛选:均一化通用文库筛选,Y187，AD 文库,Y2H Gold，BD诱饵培养液,DDO/X/A,混合,QDO/X/A,文库筛选:传统细菌文库筛选,取1ml菌液按照一定比例用LB稀释，150ul/板进行涂板，过夜培养。,将提取的质粒转化Y187，获得酵母文库。,将文库与诱饵培养液混合，进行Mating。涂筛选板，挑取阳性克隆子。,刮取平板上的菌落，提取质粒。,文库筛选:验证阳性克隆子（PCR确认）,25ul 2mix,30 PCRcycles,25ul H2O,630497 Matchmaker Insert Check PCR Mix2,文库筛选:酵母质粒提取,630467 Easy yeast Plasmid Isolation Kit,验证互作:体内验证,SEAP,630305 Mammalian Matchmaker Two-Hybrid Assay Kit 2,验证互作:体外Co-IP验证,Protein A/GAgarose beads,Matchmaker Chemiluminescent Co-IP Vector Set（630458）Matchmaker Chemiluminescent Co-IP Kit（630459）Prolabel Chemiluminescent Detection Kit（631723）,验证互作:体外Co-IP验证实验流程,PL检测,验证互作:FRET分析,632466 pDsRed-Monomer-C1 Vector,632470 pAcGFP1-C1-Vector,Clontech Matchmaker系统产品选择指南,The FRIGIDA Complex Activates Transcription of FLC,a Strong Flowering Repressor in Arabidopsis,by Recruiting Chromatin Modification Factors K Choi,J Kim,HJ Hwang,S Kim-The Plant Cell,2011-Am Soc Plant Biol Extracellular signal-regulated kinase 8(Erk8)controls estrogen-related receptor(ERR)cellular localization and inhibits its transcriptional activity M Rossi,D Colecchia,C Iavarone,A Strambi-Journal of Biological,2011 ASBMBWSB-1,a novel IL-21 receptor binding molecule,enhances the maturation of IL-21 receptor H Nara,T Onoda,M Rahman,A Araki-Cellular,2011 ElsevierFHL1 interacts with oestrogen receptors and regulates breast cancer cell growth L Ding,C Niu,Y Zheng,Z Xiong-Journal of Cellular,2011-Wiley Online LibraryRab4 interacts with the human Pglycoprotein and modulates its surface expression in multidrug resistant K562 cells C FerrndizHuertas-Journal of Cancer,2011-Wiley Online Library6.Duplicated C-class MADS-box genes reveal distinct roles in gynostemium development in Cymbidium ensifolium(Orchidaceae)SY Wang,PF Lee,YI Lee,YY Hsiao-Plant and Cell,2011-Jpn Soc Plant PhysiolProtein Interaction Domain Mapping of Human Kinetochore Protein Blinkin Reveals a Consensus Motif for Binding of Spindle Assembly Checkpoint Proteins Bub1 and BubR1 T Kiyomitsu,H Murakami-Molecular and Cellular,2011-Am Soc Microbiol,Clontech Matchmaker系统近期参考文献,主要内容,常见问题分析:酵母双杂交文库如何获得？,购买Clontech公司预制文库,用630490自己构建文库,请Takara公司提供文库构建服务,如何获得酵母双杂交文库,常见问题分析:一定要做自激活检测吗？,必须滴！,自激活检测必须做：1.需要在Y2H Gold菌株中进行。2.如果出现自激活现象需要调整AbA浓度或者调整序列。3.如果菌落生长过慢，可能是表达有毒性，需要用低拷贝质粒pGBT9(630490提供)。,常见问题分析:诱饵的大小有限制吗？,我们有文献报道的8-750个氨基酸都有成功的案例。Heery,D.M.et al.(1997)Nature 387(6634):733736.2.Schaefer B.C.,Ed.(1997)Gene Cloningand Analysis:Current Innovations,(Horizon Scientific Press),pp 1128.,常见问题分析:培养基较软或发黑？,灭菌条件控制,调整pH,购买方便包装,让培养基正常起来,：800-810-6261；/86,：,：,我们就在这里等您,Thank You！,3Q！,
