酵母双杂交系统技术介绍.ppt

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,*,*,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,*,*,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,宝日医生物技术（北京）有限公司,3/10/2025,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,Clontech,Matchmaker,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,GAL4,蛋白具有两个功能结构域,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,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！,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：,630489,630305,体外,Co-IP,验证：,630458,630459,631723,631604,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,Y2,H 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,两个载体,两个菌株,转化试剂盒,control,630489,Y187,Y2H Gold,三个载体,阳性对照，,阴性对照。,BD vector,：,pGBKT7,AD 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,诱饵载体,In Y2H Gold,文库筛选,:,诱饵载体构建及其自激活检测,Y2,H Gold,诱饵载体,文库筛选,:Mating,方法筛选,Y187,型,DDO,筛选,二倍体,Y2H Gold,a,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,Mix,Mating,QDO,筛选,文库筛选,:,均一化通用文库筛选,均一化通用型文库,高丰度与低丰度均衡,大大降低工作量,大大降低假阳性,更高的基因代表性,Normalized,Universal,Pre.library,Y187,，,AD,文库,Y2,H 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,文库培养,提取质粒,转化酵母菌,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。,Mating,筛选,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 2,mix,30 PCR,cycles,25ul H,2,O,630497,Matchmaker,Insert Check PCR Mix2,文库筛选,:,酵母质粒提取,630467,E,asy 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,Bait,AcGFP1,PL,Prey,Protein A/G,Agarose 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 ASBMB,WSB-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 Elsevier,FHL1 interacts with oestrogen receptors and regulates breast cancer cell growth,L Ding,C Niu,Y Zheng,Z Xiong-Journal of Cellular,2011-Wiley Online Library,Rab4 interacts with the human Pglycoprotein and modulates its surface expression in multidrug resistant K562,cells,C FerrndizHuertas-Journal of Cancer,2011-Wiley Online Library,6.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 Physiol,Protein 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,；,010-80720985,/,86,：,service,：,我们就在这里等您,Thank You,！,3Q,！,41,