光谱检测技术在生命科学中应用

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,第,2,章光谱检测技术在生命科学中的应用,紫外透照灯,1.,光谱范围和对应的检测仪器,400nm,650nm,Ultra Violet Visible Near Infrared,白光透照灯、普通酶标仪、荧光化学发光酶标仪、测序仪、扫描仪,显微镜,测序仪、扫描仪,凝胶图像分析系统、核酸蛋白检测仪、光密度扫描仪、激光扫描仪、荧光显微镜,分光光度计、荧光分光光度计、扫描酶标仪,紫外线,紫外线是德国物理学家里特在,1801,年发现的，一切高温物体，如太阳、弧光灯发出的光都含有紫外线。紫外线的作用主要是化学作用。紫外线有很强的荧光效应，能使很多物质发出荧光。,主要部件紫外灯管和滤光片,波长,254nm,、,302nm,、,365nm,或,2,个、,3,个波长组合一体,用于检测凝胶和膜上紫外线激发的荧光染料,EB,、,Sybr-Green,等,紫外透照仪,可见光,A,可,见,光,主要部件日光灯管和磨砂玻璃,波长可见光,用于检测凝胶和膜上的染料硝酸银、考马斯亮蓝等及放射自显影胶片,白光透照仪,主要部件,光学系统（光源、滤光片、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：,340-700nm,用于各种微孔板检测,普通酶标仪,Elx-800,酶标仪工作原理,光电倍增管,计算机,打印机,酶标板,滤光片,光源,光电效应,在光（包括不可见光）的照射下从物体发射出电子的现象叫做光电效应，发射出来的电子叫做光电子。,真空光电管：真空的玻璃泡或在内充有少量惰性气体（如氩、氖、氦等）。泡的内半壁涂有碱金属，例如钠、锂、铯，作为阴极,K,，泡内另有一阳极,A,。将光电管连在电路里，当光照射到阴极,K,时，电路里就产生电流，电流的强度取决于照射光的强度。光电管产生的电流很弱，可以用放大器把它放大。,Elx-808,酶标仪工作原理,滤光片轮,卤素灯,组成：,CCD摄像头,变焦镜、滤光片、近摄镜,暗箱,紫外透照仪,PCI图像捕捉卡,图像捕捉及分析软件,热敏打印机,电脑及显示器（OPTION）,功能：图像采集和分析,凝胶图像分析系统,主要部件,光学系统（光源、滤光片、光电倍增管）,数据处理和传输系统,微量比色杯,波长：,230,、,260,、,280,、,320,、,562,、,595,nm,用于各种生物样品检测,核酸蛋白检测仪,核酸蛋白检测仪,滤光片,光源,比色杯,光电倍增管,主要部件,光学系统（光源、单色器、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：,200-999nm,，,1nm,步进,用于各种微孔板检测,全波长扫描酶标仪,PowerWave HT,Synergy,全波长扫描酶标仪工作原理,荧光技术简介,一、荧光发生过程,荧光产生于某些分子（通常是含多聚芳香烃的碳水化合物或杂环化合物）称为荧光体或荧光染料。,荧光探针是一个荧光体设计用于定位生物样品的特异性区域或对特异性刺激因子发生反应。,荧光发生是一个,3,阶段的过程。,激发,外源的白炽灯或激光提供的能量（,hv,EX,）的光子被荧光体吸收，产生被激发的电子单峰态（,S,1,）。这个过程是荧光和化学发光的区别，化学发光的激发态是由化学反应产生的。,激发态的寿命期,激发态存在一个有限的时间（通常是,1-10x10,-9,秒）。在这时间，荧光体经历构象的改变和容易受到分子环境的相互作用的影响。这些过程有两个重要的结果。第一，,S,1,的能量部分被消耗，形成一个衰减的单峰激发态（,S,1,）即荧光发射的初始。第二，不是最初被激发的所有分子都通过荧光发射回到基态。其他的过程，象振动淬灭，荧光能量传递也在,S,1,衰减。荧光量子的产生量，取决于激发的荧光光子数量,及,吸收的光子数量的比率，是衡量荧光效率的参数。,荧光发射,能量（,hv,EM,）光子被激发，荧光体回到基态,S,0,。由于能量在激发态寿命期的部分消耗，这些光子的能量较低，因此比激发光子,hv,EX,有更长的波长。这种由于（,hv,EM,- hv,EX,）呈现的能量或波长的差异称作,Stokes,漂移。,Stokes,漂移奠定了荧光技术灵敏性的基础，因为它可以,及,激发光子在光谱上分离，而在一个较低的背景下检测发射光子。相比较而言，吸收光分光测定法在透射光检测时相对于较高水平的同一波长的光。,Emission,F,Excitation,荧光分子受到激发后释放能量的,3,种方式：发光,Emission,Excitation,F,2,F,1,Transfer,荧光分子受到激发后释放能量的,3,种方式：传递,Excitation,BH,F,Transfer,荧光分子受到激发后释放能量的,3,种方式：发热,二、荧光光谱,荧光发生的整个过程是循环的，除非荧光体在激发态不可逆转的破坏（一个重要的现象称作光漂白），同一荧光体可以反复地激发和检测。对于溶液中多原子分子，由,hv,EX,和,hv,EM,呈现的不连续的电子跃迁被一个较宽的能量光谱称作荧光激发光谱和荧光发射光谱取代。在两种或更多的荧光体同时检测时，这些光谱的带宽是非常重要的参数。除了很少的例外，一般在稀释溶液中单种荧光体的荧光激发光谱和它的吸收光谱是相同的。在同样条件下，由于在激发态寿命期激发能量的部分消耗，荧光发射光谱,及,激发波长是分离的。荧光发射强度是与在激发波长的荧光激发光谱振幅成比例的。,荧光光谱,三、荧光检测,荧光检测仪器,荧光检测系统的,4,个基本要素是：激发光源，荧光体，将激发光子,及,发射光子分离的特定波长的滤光片，检测器记录发射光子并输出，通常是电子信号或图像。,3,种主要的荧光检测仪器提供不同的数据。,荧光分光光度计：测定液体样品（,uL-mL,）,荧光显微镜：解析荧光作为二维或三维空间位置定位功能,流式细胞仪：测定流体中每个细胞的荧光，在大量样品中进行分选并识别和定量,激光扫描仪,荧光信号,荧光强度的定量取决于这些参数：吸光度,-,由,Beer-Lambert,定律定义为摩尔消光系数的产物，光径和稀释浓度，染料的荧光量子的产量，激发光源强度和仪器荧光采集效率。在稀释溶液或悬液中，荧光强度,及,这些参数成线性比例关系。当样品吸光度在,1 cm,光径超过,0.05,时，线性关系被自身吸收和内滤光效应扭曲。,通过一个大的荧光,Stokes,漂移（例如分离为,A1,和,E1,）可以容易从,Rayleigh,散射激发光（,EX,）分离出荧光发射信号（,S1,）。标记有荧光探针的生物分子通常都含有超过,1,个的荧光物，使信号分离更加复杂。另一个光信号（,S2,）可能是背景荧光或第二个荧光探针。,背景荧光,荧光检测的灵敏性会受到背景信号的很大影响，这些背景信号来自样品内在组分（自发荧光）或未结合的和非特异性结合的探针（试剂背景）。检测中可以通过选择滤光片或通过选择更长波长吸收和发射的探针来减小,E2,自发荧光对,E1,的影响。尽管将荧光检测带宽变窄增加了,E1,和,E2,间的分辨力，但是会影响总的荧光检测强度。对于细胞、组织和生物体液的自发荧光，可以采用激发光,500nm,的探针减小荧光信号失真。但是，较长波长的激发光会受到密度介质如组织的影响而发散减弱，就需要更强穿透力的激发光。,荧光的两种检测光路,直接标记 将荧光团通过化学反应直接衔接在核酸的双链或单链上,间接标记 将带有荧光团标记的,NTP,或,dNTP,通过酶反应掺入到新合成的核酸链中,荧光对核酸的修饰和标记,Physical Data for IRDye700- CDI,max = 680nm,emission = 715nm, = 190,000 L Mol,-1,cm,-1,Physical Data for IRDye800- CDI,max = 780nm,emission = 815nm, = 205,000 L Mol,-1,cm,-1,核酸染料,IRDye,结构,Hybridize,Label DNA,5 min,Fast,Convenient,Robust,Low cost,IRDye,DNA,DNA Labeling,切口平移法,随机引物掺入法,逆转录合成掺入法,Aminallyl dUTP,及,荧光团的结合,间接荧光标记,Universal Linkage System (ULS) platinum-based chemistry,氨基或硫醇基修饰的核酸可直接连接荧光团,胞嘧啶的硫化介导的荧光团标记,直接荧光标记,IRDye800,Excitation max = 778 nm,Emission max = 806 nm,E = 180,000 M,-1,cm,-1,Quantum yield = 0.34,Cy5.5,Excitation max = 675 nm,Emission max = 694 nm,E = 250,000 M,-1,cm,-1,Quantum yield = 0.28,蛋白染料,IRDye,结构,Antigen on Membrane,1,1,+,2,OR,1,+,2,1,+,2,+,3,IRDye 800 and Cy5.5 Secondary and Tertiary Labeled Antibodies,Labeled Antibodies,主要部件,光学系统（光源、滤光片、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：激发波长：,300-650nm,发射波长：,350-700nm,发光波长：,350-700nm,用于各种微孔板检测,荧光、化学发光酶标仪,Flx800,Synergy,主要部件,共聚焦光学系统（激光光源、滤光片、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：激发波长：,488,、,514,、,543,、,594,、,633nm,发射波长：,500-700nm,用于玻璃载片的生物芯片检测,ScanArray,激光共聚焦扫描仪,ScanArray,激光共聚焦原理,ScanArray,荧光激发,显微镜光路图,红外线,红外线是英国物理学家赫谢尔在,1800,年发现的。红外线最主要的作用是热作用。红外线的波长较长，因此衍射现象比较显著，穿透力很强。,主要部件,共聚焦光学系统（激光光源、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：激发波长：,685,、,785nm,发射波长：,715,、,815nm,用于测序,红外激光自动测序仪,红外激光自动测序仪,主要部件,共聚焦光学系统（激光光源、光电倍增管）,机械运动和定位系统（步进马达）,自动控制系统,数据处理和传输系统,波长：激发波长：,685,、,785nm,发射波长：,715,、,815nm,用于杂交膜检测,红外激光扫描仪,红外激光扫描仪原理,红外激光扫描仪荧光激发,IRD700,激发,IRD800,激发,荧光显微镜光路图,滤光片结构图,荧光显微镜滤光片光路图,化学发光技术简介,按光谱分类：,狭义：可见光（,400-700nm,）,广义：紫外光（,10-400nm,）,可见光（,400-700nm,）,红外光（,700-40000nm,）,光的基本知识,炽热,光或白,炽,光,(incandescence)-,因,热,而,发,光,气体激发,(gas excitation-,因,电子,或,热的激发,而,发,光,摩擦,发,光,(triboluminescence),电,激发光,(electroluminescence),荧光,(fluorescence)-,因光激,发而发光,，但光源切,断后,即不再,发,光,磷光,(phosphorescence)-,因光激,发,而,发,光，但光源切,断后,仍,会持续发光,化,学发光,(chemiluminescence-,非生物,体,把多,余,的化,学,能,转变为光能,生物,发,光,(bioluminescence),-,生物,体,利用化,学能发光,发光物质,某些荧光物质,（,例如,Luminol, C,8,H,7,N,3,O,2,）,可將化学反应中产生的能量，转化成使分子內的电子由基态,（,ground state,）,跃升到激发态,（,excited state,）,，处于激发态的分子并不穩定，將能量以光的形式释放出來；有的光波长为可见光范围，但占多数的是荧光,（,fluorescence,）,。,化学发光的原理,杂交检测中的信号物质（非放射性标记）：,核酸杂交（,Southern&Northern Blot,）,蛋白印迹（,Western Blot,）,酶联免疫（,Elisa,）,其他：,dot/slot,杂交、原位杂交、,噬菌体文库筛选、菌落文库,（,质粒文库,）,筛选,化学发光在生物学检测中的应用,一般需要一定的发光底物及发光增强剂（如,CDP-Star,，,461nm,，蓝光）,并配有各自的过氧化物酶系统。经过化学发光及增强作用，将光量放大后，在,X,片上发光自显影，将特异结合条带显示出来。,化学发光检测敏感性高，除借助特殊仪器检测外，需要在暗室进行,X,光片曝光，曝光时间要掌握适度以便得到最佳信号强度。此外，由于不同发光底物持续发光时间和强度不同，除了摸索曝光时间外还要注意在稳定持续发光时间内可以多次曝光。,。,生物学中化学发光检测原理,Luminol,（,430nm,）,CSPD,发光最强最持久：,CDP-Star,（ ,460nm,，蓝光）,Sapphire-II,发光增强剂,Emerald-II,发光增强剂,注： 一般的试剂盒中已将发光底物,及,发光增强剂预先混合为发光底物,Luminol,几种常用的发光底物及增强剂,核酸杂交：,生物素标探针酶（,AP,）标生物素抗体或,Strepavidin,CDP-Star,底物,生物素标探针酶（,AP,）标生物素抗体或,Strepavidin,CDPD,底物,生物素标探针酶（,HRP,）标生物素抗体或,Strepavidin,Enhanced Luminol,底物,荧光素标探针酶（,AP,）标抗荧光素抗体,CDP-Star,底物,荧光素标探针酶（,HRP,）标抗荧光素抗体,Enhanced Luminol,底物,地高辛标探针酶（,AP,）标抗地高辛抗体,CDPD,底物,酶（专利的耐热,AP,）标探针发光底物,几种常用的试剂盒检测原理,核酸抽提,电泳,转膜,生物素标记探针,（随机引物法,PCR,掺入法）,杂交、洗脱,酶标生物素抗体,或结合物敷育,底物敷育,化学发光检测：,X,光片压片曝光显影定影,实验流程,举 例,NEBlot Phototope Kit & Phototope-Star Chemiluminescent Detection Kit,特点：,稳定：,32,P-,标记的探针只能放置几天，而生物素标记的探针可放置较长时间,敏感：可检测哺乳动物,DNA,中的单拷贝基因,（,1pg,），,及,同位素相当,快速：整个检测程序只需,40,分钟,（,曝光时间只需,1-10,分钟，需要优化,）,发光可持续几天，可,多次曝光，以获取最佳信号强度,NEBlot Phototope Kit & Phototope-Star Chemiluminescent Detection Kit,Ambion BioDetect,TM,Nonisotopic Detection Kit,举 例,NEN,公司,化学荧光素,/,生物素标发光检测试剂盒,晶美公司,RNADetector Northern Blotting KitDNADetector Genomic Southern Blotting Kit,Tropix,（,ABI,）,Southern-Light Chemiluminescence Nucleic Acid Detection System Southern-Star Chemiluminescence Nucleic Acid Detection System,Amersham,Alkphos Labelling and Detection System,Roche,DIG Luminescent Detection Kit,其他产品,酶（,AP,）标二抗,CDP-Star,底物,酶（,AP,）标二抗,CDPD,底物,生物素标一抗酶（,HRP,）标生物素抗体或,Strepavidin,ECL,试剂,ECL,:Enhanced Chemiluminescence,，,Enhanced Luminol,底物,检测限：,DAB,显色：对,HRP,敏感，,1ng,ECL,化学发光：,15 x 12cm,CG,Chemiluminescence & Fluorescence,Reduced Budget,Higher performance,CG,2,XE,CG,2,MG,UV/white light gels,Reduced Budget,Images only,DG,GeneTools MCS,GeneTools MCSP,GeneTools/GeneDirectoryMCSPD,GG,Add Gel Analysis,From,Genius Series,GeneGnome,From,BioImaging Product Line,DigiDoc-It SystemDigital Snap Shot,BioDoc-It SystemNo Computer,GDS-8000 SystemStandard PC,Chemi SystemExpression,BioChemi SystemSensitivity,OptiChemi SystemDynamic Range,BioMicro SystemRGB Color,BioDensity SystemScanner,举 例,Chemi System,Gene and Protein Expression,Picogram Level Sensitivity,Detection 2-4 Times Longer Than Film,Cooled (-35C) CCD Camera,8-Bit Maximum Signal-to-Noise Ratio,ChemiImager 4400,ChemiDoc,MultiGenius,450,000 pixels,BioChemi System,Gene and Protein Quantitation,Mid-Femtogram Level Sensitivity,Same Detection Time as Film,Cooled (-40C) Digital CCD Camera,12, 14 and 16-Bit Acquisition,FluorChem 8800,VersaDoc 3000,440CF,1.45 million pixels,OptiChemi System,Detailed Protein Quantitation,Low-Femtogram Level Sensitivity,One Half the Detection Time as Film,Cooled (-85C) Digital CCD Camera,14 and 16-Bit Performance,Fuji LAS-1000,VersaDoc 5000,LumiImager,1.40 million pixels,FUJIFILM,LAS-1000,CCD photo element,举 例,Tropix,公司是化学发光技术专业厂家，专门从事化学发光相关仪器、应用试剂的生产和研发。在化学发光检测技术方面拥有,355,项专利。,Tropix TR717,化学发光酶标仪（,Applied Biosystems,）,1.,灵敏度,：数字,光子计数器,可检测,5,10,-21,摩尔,AP,分子，定量线性范围达,7,个数量级,采用光导纤维探头和屏蔽技术，孔间信号相互干扰小于,310,-5,，,光电倍增管检测器 波长范围：,380nm,630nm,2.,灵活性,96,孔或,384,孔微板，可编程读板方式,具有两个,RS232,接口，可兼容目前市售的全自动机械手臂,控温范围：室温以上,5,42,专利的特氟隆（,Teflon,）材质进样器，快速均匀混合反应液，保证结果精确性,3.,应用的广泛性,报告基因分析，探针杂交和免疫分析：,Tropix TR717,化学发光酶标仪特性（,Applied Biosystems,）,紫外,及,荧光光谱在蛋白质研究中的应用,主要内容,UV/vis,基础、实验,要点,、应用,Fluorescence,基础、实验,要点,、常规荧光、淬灭、荧光共振能量传递,(FRET),谱学方法,10,100 Kcal/mol,Bonds break,Light used,for vision,and,photosynthesis,Electromagnetic spectrum,Nuclear spin,vibrations,Electronic,transitions,The longer the wavelength,the lower the energy.,基本原理,1-Lambert Law,The fraction,of light absorbed by a,transparent,medium is independent of the incident intensity, and each successive layer of the medium absorbs an equal fraction of the light passing through it.,Log,10,(I,0,/I)=kl,基本原理,2-Beers Law,The amount of light absorbed is proportional to the number of molecules of the chromophore through which the light passes.,k=,c,Deviations from the Beer-Lambert law,强吸收,高浓度,噪声,及,误差,Wavelength,Absorbance,Concentration,A,B,A,B,波长选择,等吸收点,(Isosbestic point),等吸收点常作为体系中只有两种成分,(,状态,),的指示,可作为参比波长,若有等吸收点,则不需要为为每个谱作基线,单光路,双光路,双波长,二极管阵列检测器,二极管阵列光谱仪动力学实验,常规吸收光谱实验要点,样品准备,波长选择,池子选择,扫描速度选择,带宽选择,石英、玻璃、塑料,慢,快,若样品不稳定,若噪声大，分辨率低等,窄,宽,若噪声大,合适的浓度，正确的参比,紫外,/,可见光谱在蛋白质研究中的应用,浓度测定,构象变化研究,相互作用研究,蛋白质主链的紫外吸收,肽键在,250 nm,的远紫外区有较大吸收,蛋白质浓度测定,190nm, 10000 l/mol.cm,190nm,比,280nm,大,100,倍,但溶剂吸收也较大,溶剂的吸收,蛋白质中氨基酸的紫外吸收光谱,1mM,0.1mM,0.1mM,二硫键在,250 nm,附近有弱吸收,确定蛋白质的消光系数,可以根据蛋白质序列预测蛋白质的消光系数,ProtParam,:,Tyr,的吸收谱,及,pH,有关,pH titration can be used to determine,-,whether Tyr is internal or external,-,polarity of environment,max,at pH 6: 274 nm,max,at pH 13: 295 nm,环境极性的影响,Blue shift of spectra in polar solvent,Weaker absorption of Tyr in polar solvent,蛋白质构象变化研究,Absorption spectra of Poly-L-Lys HCl: random coil at pH 6.0, 25,C(bold);,-,Helix at pH 10.8, 25,C(dotted);,-strand at pH 10.8, 52,C(dashed).,蛋白质折叠,/,变性研究,荧光光谱,0,3,3,0,The Franck-Condon principle: transitions are vertical in both absorption and emission,The Franck-Condon factor is the same for absorption and fluorescence,0,0,0,1,1,0,0,3,0,6,3,0,6,0,Exceptions:,- very long lived S,1,state : emission occurs,from a different geometry.,- Reactions from the excited state.,量子产率,F,=,发射的光子数,/,吸收的光子数,荧光强度,I,F,= I,0,(1-10,-cl,) ,F,若,cl,很小，则近似：,I,F,= I,0,(cl),F,Inner filter effect,Inner filter effect,所以，实验中，,A,EX,0.1,Raman and Rayleigh Scattering,Two potential sources of background radiation are Raman and Rayleigh scattering. Both of these phenomena arise due to vibrational changes induced in molecules by incident radiation. Both can also be described as scattered light.,The Raman lines are of different frequency from the incident light and usually of longer wavelength. Rayleigh radiation is scattered light of the same frequency as the incident light.,Rayleigh Scattering,强度,及,r,6,/,4,成正比,不能通过减空白来消除,选择合适的激发波长,从比激发波长大,(10nm),处开始收谱,减少带宽,Raman scattering,因水中,O-H,收缩,(3300cm,-1,):,1/,RA,= 1/ ,EX, 0.00033,Environmental Sensitivity,Fluorophores can be affected by a large number of environmental factors including such parameters as,pH, ionic strength, non-covalent interactions, light intensity, temperature and so on,.,Both the excitation and emission wavelengths and the quantum yield can all be changed by environmental factors.,温度,荧光一般随温度上升而减弱,荧光实验需要恒温,2 ways of measuring fluorescence,Emission spectrum- excitation, constant, measure fluorescence intensity of emission against , I.e. spectrum of emitted light.,Excitation spectrum measure fluorescence intensity at different excitation , similar to absorption spectra.,Excitation spectrum,Excitation spectrum should correspond colsely with the absorption spectrum of the molecule that is responsible for the fluorescence.,Excitation spectrum reveals whether the sample is homogeneous, and whether all fluorescence features result from a single molecule.,Experiments,Spectral shifts,:,Intrinsic,Extrinsic,Fluorescence,Fluorescence quenching:,Internal, External,Fluorescence Resonance Energy Transfer (,FRET,),Rotation of molecules: fluorescence anisotropy,Fluorescence lifetime,.,Protein intrinsic fluorescence,Trp fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr),Trp is the dominant intrinsic fluorophore in proteins,Trp fluorescence,is very sensitive to its local environment,It is possible to see changes in emission spectra in response to,conformational changes, subunit association, substrate binding, denaturation,and anything that affects the local environment surronding the indole ring. Also, Trp appears to be uniquely,sensitive to collisional quenching,either by,externally added quenchers, or by,nearby groups in the protein,.,Local electric fields cause spectral shifts,Gas phase,m,m,*,solvent,Solvent can affect the ground state and excited state molecules causing spectral shift,Example: H bonding to tryptophan. Changes its absorption by about 10 nm,Fluorescence of tryptophan depends upon the dipolar nature of the solvent,Ca-parvalbumin: tryptophan is buried, 305 nm,Ca-free parvalbumin: tryptophan is exposed to solvent,Tryptophan in various solvents: hexane, trehalose glass, glycerol, water,Fluorescence spectral shifts can be very large,Tyr fluorescence,若蛋白质只含,TYR,不含,TRP,：,蛋白质变性后，荧光强度明显增强。,GFP,is isolated from the Pacific jellyfish,Aequorea victoria,and now plays central roles in biochemistry and cell biology due to its widespread use as an,in vivo,reporter of gene expression, cell lineage, protein protein interactions and protein trafficking,GFP,can be fused to another protein either N- or C-terminally. This is due to the fact that both termini of GFP appear rather flexible on the surface of the beta-can, so that GFPs structure is not significantly distorted or destroyed by the fused protein.,Ribbon diagram of the Green Fluorescent Protein (GFP) drawn from the wild-type crystal structure. The buried chromophore, which is responsible for GFPs luminescence, is shown in full atomic detail.,Green Fluorescent Protein(GFP),Different colour forms of,GFP,Additionally several different colour forms of,GFP,have been produced.,blue,cyan,green, and,yellow,FP or,BFP,CFP,GFP,and,YFP,.,Fluorescence emission spectra of equal concentrations of 1,8-ANS in ethanol:water mixtures. The labels adjacent to each curve indicate the percentage of ethanol in the solvent mixture.,Protein,Extrinsic,fluorescence,ANS,(1-anilinonaphthalene-,8-sulfonic acid),1-,苯胺基萘,-8-,磺酸,strong fluorescence enhancement,when its exposure to water is lowered,Fluorescence enhancement of 1,8-ANS, upon binding to protein. The image shows aqueous solutions of 1,8-ANS excited by ultraviolet light. Addition of protein (bovine serum albumin) to the solution in the cuvette on the left results in intense blue fluorescence. The fluorescence of uncomplexed free dye in the cuvette on the right is negligible in comparison.,1,8-ANS,is,a sensitive probe,for,partially folded intermediates,in protein-folding pathways.,Molten globule intermediates,are characterized by particularly high ANS fluorescence intensities due to the exposure of hydrophobic core regions that are inaccessible to the dye in the native structure.,ANS binding of sHSP16.5 in GdnHCl,Titration of ANS fluorescence at 475nm,Haemoglobin,is a complex of a small prosthetic group with the protein apohaemoglobin. The extrinsic fluor ANS fluoresces when added to solutions of apohaemoglobin but not with haemoglobin.,The addition of haem to the apohaemoglobin-ANS complex eliminates the fluorescence by displacing ANS.,This tells us that ANS and the haem group bind to the same site. Since ANS is only fluorescent in highly non-polar environments, the haem must bind to a highly non-polar site.,This would give valuable clues in the interpretation of X-ray diffraction patterns. E.g. you would know that a certain configuration of amino acids could be the point of interaction between haem and protein when building the structure.,Extrinsic fluorescence examples,Fluorescence Quenching,Internal quenching,due to intrinsic structural feature e.g. structural rearrangement.,External quenching,interaction of the excited molecule with another molecule in the sample or absorption of exciting or emitted light by another chromophore in sample.,Intramolecular quenching,Tyr often is quenched by nearby tryptophanes,Trp fluorescence can be quenched by neighbouring protonated acid groups. If the pK measured by monitoring trp fluorescence is the same as the pK for a known ionisable group (e.g. a carboxyl) then the group must be near the trp.,External quenching,Acrylamide, I,-, Cs,+,Stern-Volmer equation,Fluorescence quenching is a function of:,The excited state lifetime,The diffusion-limited quenching constant,The concentration of the quencher,The Stern Volmer slope:,K,SV,=,s,k,Q,F,0,/ ,F,= 1 +,t,s,k,Q,Q,Stern-Volmer equation,As a first proximation the value of K,SV,reveals the degree,Of exposure to the solvent of a Trp residue., 0M, 1M, 2M, 3M, 4M, 5M, 6M,Stern-Volmer plot at various GdnHCl concentrations,Acrylamide quenching of Trp fluorescence,Stern-Volmer constants,Fluorescence,Resonance Energy Transfer,Known as fluorescence resonance energy transfer,(FRET),or Frster energy transfer. It is the radiationless transfer of excitation energy from a donor to an acceptor. An important consequence of this transfer is that there is no emission of light by the donor. The acceptor may or may not be fluorescent. FRET is a distance-dependent interaction where the energy transfer occurs typically over a distance of,1-10nm,. The distance dependent nature of,FRET is highlighted by the fact that it is proportional to the inverse sixth power of the intermolecular separation,.,If the fluorophores (extrinsic or intrinsic) have unique locations within the protein or complex, it is possible for emission light energy from A to be absorbed by B and to be emitted as part of Bs emis