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,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,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,Western Blot,常见检测方法,化学显色法 如,DAB,显色,放射自显影法 如同位素,化学发光法 如,ECL,化学荧光法 如,IRdye680,、,IRdye780,Western Blot 常见检测方法化学显色法 如DA,化学显色法操作简便，无需暗房和显影设备，显色直观，但灵敏度较低，国外已不常见。,化学发光法（,ECL,）比化学显色法灵敏度高，可达,pg,水平。但由于是酶促反应，不同曝光时间得到的信号与样品量不成线性关系，不能做准确定量。,荧光法采用荧光检测原理，操作简单，荧光信号强弱直接反映目的蛋白量，可进行精确定量；还可以根据荧光染料的不同，用不同的荧光二抗可以在同一张膜上同时检测两种蛋白质。,不同检测方法特点,化学显色法操作简便，无需暗房和显影设备，显色直观，但灵敏度较,ECL,常见问题,暗房操作麻烦，曝光时间难以控制，无法准确定量,背景高，高浓度蛋白易出现“吹泡”现象,检测两种蛋白时，需要,Stripping,和,Re-probe,，操作复杂,胶片上无法直接观察到,Marker,ECL常见问题暗房操作麻烦，曝光时间难以控制，无法准确定量,解决方案,：,Odyssey,双色红外荧光成像系统,荧光,红外,双色,荧光准确定量、线性范围宽广,红外背景低、信噪比高,双色同时检测、同时输出,解决方案：Odyssey 双色红外荧光成像,Odyssey VS ECL,流程,ECL,Odyssey,1,跑胶,跑胶,2,转膜,转膜,3,封闭,封闭,4,结合一抗、洗涤,结合一抗、洗涤,5,结合,酶,标记二抗、洗涤,结合,红外染料,标记二抗、洗涤，成像,6,发光试剂盒,7,暗房曝光成像,Odyssey VS ECL流程ECLOdyssey1跑胶跑,Antigen on membrane,Primary antibody binds to antigen,IRDye labeled secondary antibody binds to primary antibody,荧光直接检测,INFRARED,LASER DIODE,INFRARED APD DETECTOR,ON-SCREEN Display,信号持久,不需要底物,不需要底片,/,暗室,Antigen on membranePrimary ant,荧光,准确定量,TIME,TIME,CHEMILUMINESCENCE,动态信号信号随时间变化,信号不与目标蛋白浓度成比例关系,ODYSSEY,静态信号信号不随时间变化,信号和目标蛋白浓度成比例关系,High concentration/signal,Medium concentration/signal,Low concentration/signal,Saturation,Background,0,0,0,0,INTENSITY,荧光准确定量 TIMETIMECHEMILUMINESCE,红外荧光,VS,化学发光,定量线性范围,Odyssey,能检测到,0.6pg,的蛋白（灵敏度高）,Odyssey,成像呈现一个很好的线性（定量准确）,ECL,ODYSSEY,红外荧光 VS 化学发光Odyssey能检测到0.6pg的蛋,Odyssey,系统定量的线性相关性,倍比稀释的 IRDye 800-标记的抗体.R,2,从1.5 ng to 0.8 pg 是 0.99996,极佳的线性相关性。,1.5 ng,0.8 pg,极佳的线性相关性增加了定量的准确性,Odyssey系统定量的线性相关性 倍比稀释的 IRDy,Dilutions of transferrin detected with rabbit anti-Tf primary and Alexa Fluor 680 goat anti-rabbit secondary.,Sensitivity is 250-fold higher than reported for the Bio-Rad Molecular Imager FX with fluorescent antibodies,100-fold higher than ECL(,BioTechniques 29:636-642,).,红外荧光,VS,化学发光,Dilutions of transferrin detec,红外荧光,VS,化学发光,成像效果,Odyssey,曝光秒,曝光分钟,曝光分钟,曝光分钟,红外荧光 VS 化学发光Odyssey曝光秒曝光分钟曝,红外,+,荧光,DEMO,实验步骤,Odyssey:,膜,+,一抗孵育,+,荧光标记抗体孵育,+Odyssey,扫描成像,ECL:,膜,+,一抗孵育,+,酶联二抗孵育,+,底物显色,+UVP,扫描成像,实验目的,检测培养细胞转染外源基因的表达状况,红外+荧光 DEMO实验步骤Odyssey:实验目的,Marker S1 S2 S3 S4,Marker S1 S2 S3 S4,Odyssey,结果,ECL,结果,信号强度,51 20 10 4,信号强度,302 62 30 19,（Odyssey,软件分析结果）,结论,O,dyssey,和ECL的相对灵敏度相差不大,Odyssey红外激光成像系统具有更好的线性关系和更精确的定量结果,O,dyssey,扫描图能同时看到,Marker,和目标蛋白，,ECL,看不到,Marker,10:4:2:1,稀释,Marker S1 S2 S3 S4M,红外技术优点,低背景,NIR Dyes,Biomolecules,Porphyrins,Visible,Fluorophores,PAHs,1000 nm,800,600,400,200,All other,sequencers,400-650 nm,LI-COR,700-800 nm,红外技术优点低背景NIR DyesBiomolecules,红外荧光,vs,可见荧光,膜背景信号,红外荧光 vs 可见荧光,背景更低，信噪比高，保证更好检测灵敏度,定量线性范围更宽，可达,4,个数量级，定量更准确,穿透力更强（活体成像的应用）,红外荧光优点,背景更低，信噪比高，保证更好检测灵敏度红外荧光优点,Odyssey,双色同时检测,800 nm,700 nm,Overlay,两套独立激发检测系统,700,通道：,680nm,激发,720nm,检测,800,通道：,780nm,激发,820nm,检测,Odyssey双色同时检测800 nm700 nmOver,靶蛋白+靶蛋白,核酸+结合蛋白,Marker+总蛋白,参比蛋白+靶蛋白,总蛋白+修饰化蛋白,Odyssey,双色应用,No need to strip!,No need to re-probe!,Odyssey双色应用No need to strip!No,双通道同时检测,Target protein,Anti-rabbit 800 channel,Normalizer target,Anti-mouse 700 channel,*,Two-color detection requires primary antibodies from different hosts,双通道同时检测Target proteinNormalize,双色,ERK,蛋白的磷酸化,Anti-EGFR and anti-phospho-EGFR antibody specificity in A431 cells,Single color images(B and C)can be overlaid(A)to show both total protein and phosphorylated protein.,The mobility shift caused by phosphorylation is visible(A)as indicated by the red bands above the yellow bands.(yellow indicates overlapping red and green signals).,双色ERK蛋白的磷酸化Anti-EGFR and anti,双色,EMSA,Binding of T7 RNAP to two DNA fragments containing the T7 promoter sequence,labeled with either IRDye 700(red)or IRDye 800(green).,700 and 800 Overlay,700 nm Channel,800 nm Channel,DNA-Protein Complex,Unbound DNA IRDye 800,Unbound DNA IRDye 700,双色EMSABinding of T7 RNAP to t,Odyssey,双色检测的优越性,双色输出，两个目标基因同时检测，减少了工作量，而且杂交条件均等，便于两个数据的对比,杂交结果对比输出，结论更加直观，一目了然,可进行准确的量化分析,Odyssey双色检测的优越性双色输出，两个目标基因同时检测,In-Cell Western Assay,The Odyssey,In-Cell Western(ICW)Assay i,s a high-throughput approach to simultaneously detect and quantify two separate proteins directly within cells.,In-Cell Western AssayThe Odyss,In-Cell Western Assay,In-Cell Western Assay,细胞培养,Culture cells to confluency in 96-well or 384-well plates,处理细胞,Treat cells(inhibitor,stimulator,etc),固定细胞,Fix cells(,3.7%,formaldehyde,methanol or Prefer),透化细胞,Permeabilize cells(,0.1%,Triton-X 100),Incubate with IRDye,secondary antibodies,(e.g.:IRDye,680 and IRDye,800CW),Incubate with 2 primary antibodies,Incubate with 1 primary antibody,Incubate with IRDye,secondary antibody and,a DNA stain(e.g.:To-Pro-3),Odyssey,系统扫描,Scan plate directly and analyze on the LI-COR Infrared Imaging Systems.,In-Cell Western,工作流程,细胞培养 Culture cells to confluen,Membrane Westerns vs ICW,Membrane Westerns vs ICW,EGF,对,ERK,磷酸化水平的调控,700 nm(Total ERK),800 nm(Phospho-ERK),Composite Image Overlay,EGF Concentration,-,0,200,400,600,800,1000,1200,1400,1600,1800,0,0.2,0.4,0.8,1.6,3.12,6.25,12.5,25,50,100,%Induction of ERK,Phosphorylation,Conc