蛋白质及其标记演示幻灯片.ppt

Advanced Bioanalytical Chemistry Fall, 2016 朱 志 卢嘉锡 542 zhuzhi@xmu.edu.cn 第六节课 生物分子的化学标记和探针技术 2016年11月3日 ;生物分子的化学标记和探针技术;Some of the common bioconjugate designs often used for life science applications include: (A) streptavidin–enzyme conjugate (B) an immobilized affinity ligand on a particle (C) an oligo molecular beacon probe containing two fluorescent labels or a fluor and a quencher at each end (D) fluorescently labeled streptavidin (E) an affinity ligand attached to a surface (F) a biotinylated enzyme (G) an antibody–enzyme conjugate (H) a fluorescently labeled antibody (I) a biotinylated antibody (J) a biotinylated oligo probe (K) an antibody–drug conjugate (L) a gadolinium chelate-modified dendrimer containing folate molecules for targeting.;;Localization of Molecules with Fluorescence Microscopy;Fluorescence Applications in Biology;Examples of Fluorescent Molecules;; Spectral observables for fluorescence sensing. From left to right: Intensity, intensity ratio, anisotropy, time-domain lifetime, and phase-modulation lifetime. ;氨基的反应（赖氨酸侧链与N末端氨基）; 质子化率的计算： lg([质子受体]/[质子供体])=pH-pKLys ε-+NH3 pK=10.5 求ε- +NH3 在pH9.5及pH=11时的质子化%。;典型的NHS酯类荧光标记试剂;典型的NHS酯类荧光标记试剂;氨基的反应（赖氨酸侧链与N末端氨基）;;巯基的反应（半胱氨酸侧链） 与卤乙酰胺反应;巯基的反应（半胱氨酸侧链） 与顺丁烯二酰亚胺（maleimide）反应;氨基与羧基的反应Carbodiimides (–N=C=N–) 碳酰二亚胺法;EDC (1-(3-二甲氨基丙基)-3-乙基碳二亚胺); Mechanism;EDC plus Sulfo-NHS;DCC(二环己基碳二亚胺常溶有机用于有机合成);Bioconjugate Techniques, Third Edition?3rd Edition by?Greg T. Hermanson?(Author);Biological Labeling with Green Fluorescent Protein (GFP);双砷染料-四半胱氨酸体系;ReAsH (a phenoxaine derivative);双砷染料-四半胱氨酸重组噬菌体的构建及 应用;; The term ‘‘click chemistry’’, originally brought up by K. Barry Sharpless at The Scripps Research Institute in 2001.;Azide/Alkyne Cycloaddition;Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition;Staudinger-Bertozzi反应;Staudinger ligation reaction scheme (azide-phosphine conjugation). Phosphine-activated proteins or labeling reagents react with azide-labeled target molecules to form