硕士研究生课-生物化学与分子生物学课件.ppt

1、1生物系统中目标蛋白分子的定位、分离与纯化、分析与鉴定生物系统中目标蛋白分子的定位、分离与纯化、分析与鉴定蛋白质实验研究技术进展蛋白质实验研究技术进展2生物系统中目标蛋白分子的定位、分离与纯化、分析与鉴定生物系统中目标蛋白分子的定位、分离与纯化、分析与鉴定蛋白质实验研究技术进展蛋白质实验研究技术进展3生物系统中目标蛋白分子的定位生物系统中目标蛋白分子的定位三个关键问题三个关键问题:v目标蛋白分子病理条件下的位置变化目标蛋白分子病理条件下的位置变化v避免假阳性避免假阳性v提高检测灵敏度提高检测灵敏度4相关技术相关技术：免疫细胞化学技术放射自显影技术X-射线显微摄影术普通光学显微镜技术透射电子显微

2、镜技术扫描电子显微镜技术荧光显微镜技术共焦激光扫描显微镜技术5免疫细胞化学技术免疫细胞化学技术直接法（直接法（direct method）间接法（间接法（indirect method）荧光素(fluorescein,FITC,TRITC)辣根过氧物酶(horseradish peroxidase,HP)磷酸酶(phosphatase)AntigenAntibodyFirst antibodySecond antibody-2Second antibody-16过氧化物酶过氧化物酶-抗过氧化物酶复合物法抗过氧化物酶复合物法,PAP（peroxidase-antiperoxidase compl

3、ex method,PAP）First antibodySecond antibodyAntigenHorseradish peroxidae antibodyHorseradish peroxidae7标记亲和素标记亲和素-生物素法（生物素法（Labelled Avidin-Biotin method,LAB）direct methodindirect methodAntibodySecond antibodyAntigenHorseradish peroxidaeFirst antibodyAntigenAvidinHorseradish peroxidaeBiotinBiotinAvid

4、in8桥连亲和素桥连亲和素-生物素法生物素法,BABbridged avidin-biotin method亲和素亲和素-生物素生物素-过氧化物酶复合物法过氧化物酶复合物法,ABCavidin-biotin-peroxidase complex methodFirst antibodyAntigenAvidinHPBiotinFirst antibodyAntigenAvidinHPBiotinSecond antibodySecond antibody9放射自显影技术放射自显影技术(autoradiography,ARG)制备放射性同位素标记探针把放射性探针注入动物体内或加入生长在玻片上的细

5、胞的培养液中一定时间后，杀死动物取材制成切片或从培养液中取出玻片在黑匣中,切片或玻片与X-光片或涂有核乳胶的感光干板相贴感光显影、定影处理放射性同位素信号定位图像目标分子定位情况10 X-X-射线显微摄影术射线显微摄影术（X-ray microphotography）利用不同元素的原子对X射线吸收程度的差异，对组织标本内的某种特殊元素成分某种特殊元素成分进行定性定位或半定量定位分析。11RedOrangeYellowGreenCyanBlueVioletThermal radiationEmission lightFluorescenceExcitation lightUltraviolet（

6、250400nm）Generation of fluorescence12312Fluorescent dyeNameExcitation light,nm Emission light,nmUsagePrimulin(樱草素)360400500Cell/BacteriaThioflavine(硫代黄素)380420550Cell/BacteriaCoumarin(香豆素)360450Cell/BacteriaFluorescein(荧光素)405480Cell/BacteriaAuramine(金胺,槐黄)435490590Cell/BacteriaFluorescein diacetica

7、te(二乙酸荧光素)460610Cell survival13Fluorescent dye,continueNameExcitation light,nm Emission light,nmUsageRhodamine 123(罗丹明 123)560540660MitochondrionJC-1(5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidalyl carbocynine iodide)490527(low concentration or low poten)/590(high concentration or high poten)Mit

8、ochondrionDAMP N-(3-(2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methy-lamine,dihydrochloride346LysosomeNeutral red(中性红)541640LysosomeDiOC6(dihexyloxacarbocyanine iodide)459584Endoplasmic reticulumNBD Ceramide(6-(N-(7-nitrobenz-2-oxa-1,3-diazolyl)-4-amino)caproyl)sphingosine)464532Golgi complex

9、14Fluorescent dye,continueNameExcitation light,nm Emission light,nmUsageCoumarin(香豆素)360450Label AntibodyFluorescein isothiocynate,FITC(异硫氰酸荧光素)488490610Label AntibodyPhycoerythrobilin(藻红素)488570Label AntibodyDuochrome(二铬黄)488630Label AntibodyTexas red(德克撒斯红)600630Label Antibody15Fluorescent dye,con

10、tinue NameEx.nm Em.nmUsageHoechst 33258(赫斯特 33258)360505DNAMithramycin(光神霉素)457570DNAEthidium bromide(溴化乙啶)488610DNAPropidium iodide(碘化丙啶)488620DNAAcridine orange405530640DNA or RNAPyroniny(派若宁丫)488580RNA16Fluorescent dye,continue NameEx.nm Em.nmUsageCoriphosphine(沉香硫化氢)458470660Lipids17Fluorescent

11、dye,continue NameEx.nm Em.nmUsageIndo-1355405Ca2+(high)Fluo-3490525Ca2+Calcium green-5N506531Ca2+(high)Rhod-2550575Ca2+(high)Calcium orange554575Ca2+(high)Calcium Crimson588611Ca2+(high)Fura-Red488660Ca2+(high)18Fluorescent dye,continue(Usage:pH)NameEx.peak,Diex.rate,nm Em.peak,Diem.rate,nmBCECF,ran

12、ge:6.47.4437/508,490/440520/531,530/640Cl-NERF503/514,488/514540/539CMHC327/372,372/327470/470COFDA450/495,490/450520/520,520DHPN380/405,420/360453/483,512/455DHPDS405/458,458/405465/500,500/465DM-NERF497/510,488/514527/5354-MU330/360,365/334450,450/550Pyranine400/465,450/400510/510,465/510Quene-1390530SNAFL-2510/546,540/480539/630,630/520SNAFL-calcein506/538535/620SNARF-1,range:6.38.6546/575,590/500588/673,636/568SNARF-calcein506(533)/585580/630