碩士研究生課現(xiàn)代分子生物學(xué)蛋白質(zhì)研究進(jìn)展李恩民

1、1生物系統(tǒng)中目標(biāo)蛋白分子的定位、分離與純化、分析與鑒定生物系統(tǒng)中目標(biāo)蛋白分子的定位、分離與純化、分析與鑒定蛋白質(zhì)實(shí)驗(yàn)研究技術(shù)進(jìn)展蛋白質(zhì)實(shí)驗(yàn)研究技術(shù)進(jìn)展2生物系統(tǒng)中目標(biāo)蛋白分子的定位、分離與純化、分析與鑒定生物系統(tǒng)中目標(biāo)蛋白分子的定位、分離與純化、分析與鑒定蛋白質(zhì)實(shí)驗(yàn)研究技術(shù)進(jìn)展蛋白質(zhì)實(shí)驗(yàn)研究技術(shù)進(jìn)展3生物系統(tǒng)中目標(biāo)蛋白分子的定位生物系統(tǒng)中目標(biāo)蛋白分子的定位三個(gè)關(guān)鍵問(wèn)題三個(gè)關(guān)鍵問(wèn)題: :v目標(biāo)蛋白分子病理?xiàng)l件下的位置變化目標(biāo)蛋白分子病理?xiàng)l件下的位置變化v避免假陽(yáng)性避免假陽(yáng)性v提高檢測(cè)靈敏度提高檢測(cè)靈敏度4相關(guān)技術(shù)相關(guān)技術(shù)：免疫細(xì)胞化學(xué)技術(shù)放射自顯影技術(shù)X-射線顯微攝影術(shù)普通光學(xué)顯微鏡技術(shù)透射電子

2、顯微鏡技術(shù)掃描電子顯微鏡技術(shù)熒光顯微鏡技術(shù)共焦激光掃描顯微鏡技術(shù)5免疫細(xì)胞化學(xué)技術(shù)免疫細(xì)胞化學(xué)技術(shù)直接法（直接法（direct method）間接法（間接法（indirect method）熒光素 (fluorescein, FITC, TRITC)辣根過(guò)氧物酶 (horseradish peroxidase, HP)磷酸酶 (phosphatase)AntigenAntibodyFirst antibodySecond antibody-2Second antibody-16過(guò)氧化物酶過(guò)氧化物酶-抗過(guò)氧化物酶復(fù)合物法抗過(guò)氧化物酶復(fù)合物法, PAP（peroxidase-antiperoxid

3、ase complex method, PAP）First antibodySecond antibodyAntigenHorseradish peroxidae antibodyHorseradish peroxidae7標(biāo)記親和素標(biāo)記親和素-生物素法（生物素法（Labelled Avidin-Biotin method, LAB）direct methodindirect methodAntibodySecond antibodyAntigenHorseradish peroxidaeFirst antibodyAntigenAvidinHorseradish peroxidaeBioti

4、nBiotinAvidin8橋連親和素橋連親和素-生物素法生物素法, BABbridged avidin-biotin method親和素親和素-生物素生物素-過(guò)氧化物酶復(fù)合物法過(guò)氧化物酶復(fù)合物法, ABCavidin-biotin -peroxidase complex methodFirst antibodyAntigenAvidinHPBiotinFirst antibodyAntigenAvidinHPBiotinSecond antibodySecond antibody9放射自顯影技術(shù)放射自顯影技術(shù)(autoradiography, ARG)制備放射性同位素標(biāo)記探針把放射性探針注入

5、動(dòng)物體內(nèi)或加入生長(zhǎng)在玻片上的細(xì)胞的培養(yǎng)液中一定時(shí)間后，殺死動(dòng)物取材制成切片或從培養(yǎng)液中取出玻片在黑匣中, 切片或玻片與X-光片或涂有核乳膠的感光干板相貼感光顯影、定影處理放射性同位素信號(hào)定位圖像目標(biāo)分子定位情況10 X-X-射線顯微攝影術(shù)射線顯微攝影術(shù)（ X-ray microphotography） 利用不同元素的原子對(duì)X射線吸收程度的差異，對(duì)組織標(biāo)本內(nèi)的某種特殊元素成分某種特殊元素成分進(jìn)行定性定位或半定量定位分析。11RedOrangeYellowGreenCyanBlueVioletThermal radiationEmission lightFluorescenceExcitation

6、 lightUltraviolet （250400nm）Generation of fluorescence12312Fluorescent dyeNameExcitation light, nm Emission light, nmUsagePrimulin (櫻草素)360400500Cell/BacteriaThioflavine (硫代黃素)380420550Cell/BacteriaCoumarin (香豆素)360450Cell/BacteriaFluorescein (熒光素)405480Cell/BacteriaAuramine (金胺, 槐黃)435490590Cell/Ba

7、cteriaFluorescein diaceticate (二乙酸熒光素)460610Cell survival13Fluorescent dye, continueNameExcitation light, nm Emission light, nmUsageRhodamine 123 (羅丹明 123)560540660MitochondrionJC-1(5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidalyl carbocynine iodide)490527(low concentration or low poten) /590(hig

8、h concentration or high poten)MitochondrionDAMP N-(3-(2,4-dinitrophenyl) amino)propyl)-N-(3-aminopropyl) methy-lamine, dihydrochloride346LysosomeNeutral red (中性紅)541640LysosomeDiOC6 (dihexyloxacarbocyanine iodide)459584Endoplasmic reticulumNBD Ceramide (6-(N-(7-nitrobenz-2-oxa-1,3-diazolyl)-4- amino

9、) caproyl)sphingosine)464532Golgi complex14Fluorescent dye, continueNameExcitation light, nm Emission light, nmUsageCoumarin (香豆素)360450Label AntibodyFluorescein isothiocynate, FITC (異硫氰酸熒光素)488490610Label AntibodyPhycoerythrobilin (藻紅素)488570Label AntibodyDuochrome (二鉻黃)488630Label AntibodyTexas re

10、d (德克撒斯紅)600630Label Antibody15Fluorescent dye , continue 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 (派若寧丫)488580RNA16Fluorescent dye , continue NameEx.

11、nm Em. nmUsageCoriphosphine (沉香硫化氫)458470660Lipids17Fluorescent 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 dy

12、e, continue (Usage: pH)NameEx.peak, Diex.rate, nm Em.peak, Diem.rate, nmBCECF, range: 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, 450/550Pyranine400/4