蛋白相互作用酵母双杂交.pptxVIP

蛋白相互作用与酵母双杂交 Protein-protein Interaction Yeast Two-hybrid ;;Protein-protein Interaction;蛋白质相互作用研究技术;Slide 5;噬菌体展示技术的原理;biotin;Bind to Streptavidin;Wash to remove unbound phage particles.;Elute bound phage;Amplify eluted phage Repeat selection Analyze a) ELISA b) Specificity c) Sequencing d) Affinity e) Activity ;Slide 12;Slide 13;RIGI;Cross-linking;;;;酵母双杂交系统的原理;;三个启动子，四个报告基因;;Restriction Map and Multiple Cloning Site (MCS) of pGBKT7;Restriction Map and Multiple Cloning Site (MCS) of pGADT7.;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;Transformation of Competent Yeast Cells: 1. 加入质粒DNA 100-500ng 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，10000rpm 15s 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（GAL UAS）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。;Transcription factors;You should demonstrate that your bait protein is not toxic when expressed in yeast. If your bait is toxic to the yeast cells, both solid and liquid cultures will grow more slowly. If expression of your bait protein does have toxic effects, you may wish to switch to a vector (such as pGBT9) that has a lower level of expression. 1. Materials: ? Y2HGold competent cell ? SD/–Trp agar plates ? SD/–Trp broth 2. Transform 100 ng of the following vectors: ? pGBKT7 (empty) ? pGBKT7 + cloned bait gene 3. Spread 100 μl of 1/10 and 1/100 dilutions of your transformation mixtures onto SD/–Trp. 4. Grow at 30°C for 3–5 days: Note : If your bait is toxic, you may notice that colonies containing your bait vector are significantly smaller than colonies containing the empty pGBKT7 vector.;;Slide 38;(一) 酵母单杂交系统(one-hybrid system);酵母单杂交系统原理 ;(二)