蛋白相互作用&酵母双杂交.pptx

蛋白相互作用与酵母双杂交

Protein-proteinInteraction
&
YeastTwo-hybrid
Protein-proteinInteraction蛋白质相互作用研究技术Slide5噬菌体展示技术的原理biotinBindtoStreptavidinWashtoremoveunboundphageparticles.EluteboundphageAmplifyelutedphage

Repeatselection

Analyze
a)ELISA
b)Specificity
c)Sequencing
d)Affinity
e)Activity
Slide12Slide13RIGICross-linking酵母双杂交系统的原理三个启动子，四个报告基因RestrictionMapand
MultipleCloningSite(MCS)ofpGBKT7RestrictionMapand
MultipleCloningSite(MCS)ofpGADT7.基因序列克隆基因序列克隆酵母质粒双酶切线性化融合酶融合转化大肠杆菌感受态并测序测序正确的质粒转化酵母TransformationofCompetentYeastCells:
1.加入质粒DNA100-500ng
鲑鱼精DNA(变性的，10mg/ml) 5ul
加入感受态细胞,轻弹混匀 50ul
加入PEG/LiAc,轻弹混匀 500ul
2.30℃水浴，每10轻弹混匀 30min
3.加入DMSO,轻弹混匀 20ul 4.42℃水浴，每5min轻弹混匀 15min
5.离心，弃上清，10000rpm15s
6.用0.9%NaCl重悬 1ml
7取适量菌液涂布在相应的筛选培养基上。

涂布单缺培养基挑选发育良好菌落mating并随后二缺四缺培养基筛选一般情况下，单独的BD可以与GAL4上游活化序列（GALUAS）结合，但不能引起转录。然而，将一段具有转录激活活性的转录因子基因构建到BD载体上，若其表达产生的BD单独与UAS结合也可以引起下游报告基因的转录，那么就称之为酵母双杂中的自激活现象。TranscriptionfactorsYoushoulddemonstratethatyourbaitproteinisnottoxicwhenexpressedinyeast.Ifyourbaitistoxictotheyeastcells,bothsolidandliquidcultureswillgrowmoreslowly.
Ifexpressionofyourbaitproteindoeshavetoxiceffects,youmaywishtoswitchtoavector(suchaspGBT9)thathasalowerlevelofexpression.
1.Materials:
•Y2HGoldcompetentcell
•SD/–Trpagarplates
•SD/–Trpbroth
2.Transform100ngofthefollowingvectors:
•pGBKT7(empty)
•pGBKT7+clonedbaitgene
3.Spread100μlof1/10and1/100dilutionsofyourtransformationmixturesontoSD/–Trp.
4.Growat30°Cfor3–5days:
Note:Ifyourbaitistoxic,youmaynoticethatcoloniescontainingyourbaitvectoraresignificantlysmallerthancoloniescontainingtheemptypGBKT7vector.Slide38(一)酵母单杂交系统(one-hybridsystem)酵母单杂交系统原理(二)反向酵母双杂交系统(reverseyeasttwo-hybridsystem)(二)反向酵母双杂交系统（reverseyeasttwo-hybridsystem）反向双杂交系统基本原理示意图Slide48存在问题与下一步计划Thanksforyourattention！