科研设计ppt.pptVIP

酵母双杂交 Surface Plasmon Resonance (SPR，表面等离子体共振) One interaction partner (Y) is bound to the metal film while the other (red balls) partner Is injected over the surface. Amount of bound protein is quantified based upon angle of reflected light. Real time association and dissociation of a protein-protein interaction can be quantified. Surface Plasmon Resonance (SPR) F?rster/Fluorescent Resonance Energy Transfer (FRET) a non-radiative, dipole-dipole coupling process, transfer energy from an excited donor fluorophore to an acceptor fluorophore in very close proximity (typically within 10nm) FRET 研究结果 2. PAX3与SOX10相互作用的免疫共沉淀检测 Zhang H, et al. Hum Genet, 2012,131:491-503. CAT assay： acetylated non-acetylated chloremphenicol are cheched by TLC CAT assay： ELISA ?-gal (?-galactosidase): E. coli enzyme (encoded by lacZ) that hydrolyzes sugars such as lactose Many assay formats: colorimetric, fluorescent, chemiluminescent SEAP (secreted alkaline phosphatase): Secreted outside the cell (can assay sample repeatedly and non-destructively by sampling culture medium) This protein is quantified directly by measuring the enzyme activity in the supernatant of the culture medium. Fluorescence and chemiluminescence assays are available for detection. Luciferase: Firefly (Photinus pyralis) luciferase Sea pansy (Renilla reniformis) luciferase Firefly luciferase produces light by ATP-dependent Photinus pyralis Renilla reniformis GFP: Green Fluorescent Protein Derived from jellyfish Aequorea victoria Autofluorescent upon UV irradiation (doesn’t require cofactors or substrates) Retains activity in presence of heat, denaturants, detergents, most proteases Allows for non-invasive monitoring of gene expression in living tissues 研究方法 荧光素酶活性检测（Luciferase activity assay） 研究结果 A B MITF promoter MITF promoter PAX3 SOX10 研究结果 E248fs突变对野生SOX10蛋白产生显性负效应作用 课程提纲 蛋白质加标签 (Tag); 定点突变； 转染; 亚细胞定位； Reporter assay； DNA-蛋白质相互作用； 蛋白质相互作用； 蛋白质半衰期。 亚细胞定位 subcellular localization 免疫荧光