筛选平台85中国药科大学药物筛选课件.pptVIP

d. Yeast Expression As yeast offers null background for human receptors, human GPCRs along with appropriate mammalian G-proteins can be expressed in yeast coupled to the pheromone signaling pathway (信息素传导途径) to screen for agonists and antagonists. GPCRs (G-protein coupled receptors): serotonin receptors, dopamine receptors, adrenergic receptors. The mating factor receptor in S. cerevisiae is called Ste2 and is similar in structure to mammalian GPCRs. Mammalian GPCR can be used to replace Ste2, so that GPCR can signal through the mating factor signaling pathway when activated by the GPCR agonist. e. Two-hybrid Yeast system Protein-protein expression 2、Mammalian Cell-Based Assays Cell-based assays differ from more traditional screening enzyme- or antibody-based assays in that the use of live cells requires special considerations. a/ free of mycoplasma（支原体）; b/ cells from frozen stock should be viable without alteration in the growth curve; c/ target protein should be expressed at a high enough level in the cells; d/ little fluctuation in replicates ……. Cell-based assays will take several days before initiation of the assays. The readouts of homogeneous format are radioactive, luminescence, or fluorescence. 1). Radioactive Assays Cell-based radioactive homogeneous assays have been used for functional assays and receptor-ligand assays. Receptor binding assays Receptor binding assays with membrane receptors can also be assays with whole cells, either adherent or suspension cells, with a radioligand. GTP-?-S binding assays G-protein activation can be assessed by measuring the agonist induced binding of a nonhydrolyzable GTP analog [35S]GTPγ S to cells: SPA FlashPlates Fluorescence intensity assays Fluorogenic assays (CBP + PPAR?) Fluorescence quench assays (CypA + L) Fluorogenic assays (CBP + PPAR?) 5/ CPA 顺十八碳四烯酸 ?max (410 nm) PPAR? Ligand CPA PPAR? ?max (410 nm) Fluorescence quench assays (CypA + Ligand) 随着DDDC838浓度增大，CypA荧光值下降，实验中，CypA浓度 保持为