高级生物化学 1.pptVIP

Two-Hybrid Library Screening Using Yeast Mating After 20 hr, check a drop of the culture under a phase contrast microscope (40X). If zygotes are present, continue to Step 8, if not, allow mating to continue; incubate for an additional 4 hr. Two-Hybrid Library Screening Using Yeast Mating Centrifuge to pellet the cells (1,000 g for 10 min). Meanwhile, rinse the 2L flask twice with 50 ml 0.5xYPDA (with 50 μg/ml kanamycin), combine the rinses, and use this to resuspend the pelleted cells. Centrifuge to pellet the cells (1,000 g for 10 min) and discard the supernatant. Resuspend all pelleteted cells in 10 ml of 0.5X YPDA/Kan liquid medium. Measure the total volume of cells+ medium. From the mated culture, spread 100 μl of 1/10, 1/100, 1/1,000, and 1/10,000 dilutions on each of the following 100 mm agar plates and incubate at 30°C for 3–5 days Two-Hybrid Library Screening Using Yeast Mating SD/–Trp SD/–Leu SD/–Leu/–Trp (DDO) Plate the remainder of the culture, 200 μl per 150 mm on DDO/X/A (50–55 plates). Incubate at 30°C for 3–5 days. Patch out all the blue colonies that grew on DDO/X/A onto higher stringency QDO/X/A agar plates using a flat sterile toothpick or yellow pipette tip Conformation of Positive Interactions Rescue of the Prey Plasmid False Positive: Genuine Positive: Sequence Analysis of a Genuine Positive Biochemical Methods to Confirm Positive Interactions Downstream Analysis Quantitative test for interactions Confirm interactions via immunoprecipitation assays Introduction GAL 4 AD:the Gal 4 transciptional activation domain GAL 4 BD:the Gal 4 DNA-binding domain This technology can be used to : identify novel protein interactions confirm putative interactions define interacting domains The map of pGBKT7-53 DNA-BD control plasmid The map of pGADT7-T AD control plasmid Y2H Gold (Mating Partner) reporter gene coustructs Y187 (library Host Strain) reporter gene constructs The entire Matchmaker screening process Step 1.Control Exepriments Step 2.Clone and t