Odyssey紅外激光成像系統(tǒng) —蛋白研究的多功能平臺(tái)【知識(shí)薈萃】

1、Odyssey紅外激光成像系統(tǒng) 蛋白研究的多功能平臺(tái),Western Blot 常見檢測(cè)方法,化學(xué)顯色法 如DAB顯色 放射自顯影法 如同位素 化學(xué)發(fā)光法 如 ECL 化學(xué)熒光法 如IRdye680、IRdye780 ,化學(xué)顯色法操作簡(jiǎn)便，無(wú)需暗房和顯影設(shè)備，顯色直觀，但靈敏度較低，國(guó)外已不常見。 化學(xué)發(fā)光法（ECL）比化學(xué)顯色法靈敏度高，可達(dá)pg水平。但由于是酶促反應(yīng)，不同曝光時(shí)間得到的信號(hào)與樣品量不成線性關(guān)系 ，不能做準(zhǔn)確定量。 熒光法采用熒光檢測(cè)原理，操作簡(jiǎn)單，熒光信號(hào)強(qiáng)弱直接反映目的蛋白量，可進(jìn)行精確定量；還可以根據(jù)熒光染料的不同，用不同的熒光二抗可以在同一張膜上同時(shí)檢測(cè)兩種蛋白質(zhì)。,

2、不同檢測(cè)方法特點(diǎn),ECL常見問(wèn)題,暗房操作麻煩，曝光時(shí)間難以控制，無(wú)法準(zhǔn)確定量 背景高，高濃度蛋白易出現(xiàn)“吹泡”現(xiàn)象 檢測(cè)兩種蛋白時(shí)，需要Stripping和Re-probe，操作復(fù)雜 膠片上無(wú)法直接觀察到Marker,解決方案：Odyssey,雙色紅外熒光成像系統(tǒng) 熒光 紅外 雙色 熒光準(zhǔn)確定量、線性范圍寬廣 紅外背景低、信噪比高 雙色同時(shí)檢測(cè)、同時(shí)輸出,Odyssey VS ECL流程,熒光直接檢測(cè),INFRARED LASER DIODE,INFRARED APD DETECTOR,ON-SCREEN Display,信號(hào)持久 不需要底物 不需要底片/暗室,熒光準(zhǔn)確定量,TIME,TIM

3、E,CHEMILUMINESCENCE 動(dòng)態(tài)信號(hào)信號(hào)隨時(shí)間變化 信號(hào)不與目標(biāo)蛋白濃度成比例關(guān)系,ODYSSEY 靜態(tài)信號(hào)信號(hào)不隨時(shí)間變化 信號(hào)和目標(biāo)蛋白濃度成比例關(guān)系,High concentration / signal,Medium concentration / signal,Low concentration / signal,0,0,0,0,INTENSITY,紅外熒光 VS 化學(xué)發(fā)光 定量線性范圍,Odyssey能檢測(cè)到0.6pg的蛋白（靈敏度高） Odyssey成像呈現(xiàn)一個(gè)很好的線性（定量準(zhǔn)確）,ECL,ODYSSEY,Odyssey系統(tǒng)定量的線性相關(guān)性,倍比稀釋的 IRDye

4、800-標(biāo)記的抗體. R2 從1.5 ng to 0.8 pg 是 0.99996, 極佳的線性相關(guān)性。,1.5 ng,0.8 pg,極佳的線性相關(guān)性增加了定量的準(zhǔn)確性,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 antibod

5、ies, 100-fold higher than ECL (BioTechniques 29:636-642).,紅外熒光 VS 化學(xué)發(fā)光,紅外熒光 VS 化學(xué)發(fā)光 成像效果,Odyssey,曝光秒,曝光分鐘,曝光分鐘,曝光分鐘,紅外+熒光 DEMO,實(shí)驗(yàn)步驟,Odyssey: 膜+一抗孵育+熒光標(biāo)記抗體孵育+Odyssey掃描成像 ECL: 膜+一抗孵育+酶聯(lián)二抗孵育+底物顯色+ UVP掃描成像,實(shí)驗(yàn)?zāi)康?檢測(cè)培養(yǎng)細(xì)胞轉(zhuǎn)染外源基因的表達(dá)狀況,Marker S1 S2 S3 S4,Marker S1 S2 S3 S4,Odyssey結(jié)果,ECL結(jié)果,信號(hào)強(qiáng)度 51 20 10 4,信號(hào)強(qiáng)度

6、302 62 30 19,（Odyssey軟件分析結(jié)果）,結(jié)論,Odyssey和ECL的相對(duì)靈敏度相差不大 Odyssey紅外激光成像系統(tǒng)具有更好的線性關(guān)系和更精確的定量結(jié)果 Odyssey掃描圖能同時(shí)看到Marker 和目標(biāo)蛋白，ECL看不到 Marker,10:4:2:1 稀釋,紅外技術(shù)優(yōu)點(diǎn)低背景,紅外熒光 vs 可見熒光 膜背景信號(hào),背景更低，信噪比高，保證更好檢測(cè)靈敏度 定量線性范圍更寬，可達(dá)4個(gè)數(shù)量級(jí)，定量更準(zhǔn)確 穿透力更強(qiáng)（活體成像的應(yīng)用）,紅外熒光優(yōu)點(diǎn),Odyssey雙色同時(shí)檢測(cè),800 nm,700 nm,Overlay,兩套獨(dú)立激發(fā)檢測(cè)系統(tǒng) 700通道：680nm激發(fā) 720

7、nm檢測(cè) 800通道：780nm激發(fā) 820nm檢測(cè),靶蛋白+靶蛋白 核酸+結(jié)合蛋白 Marker + 總蛋白 參比蛋白 + 靶蛋白 總蛋白 + 修飾化蛋白,Odyssey雙色應(yīng)用,No need to strip!,No need to re-probe!,雙通道同時(shí)檢測(cè),Target protein Anti-rabbit 800 channel,Normalizer target Anti-mouse 700 channel,* Two-color detection requires primary antibodies from different hosts,雙色ERK蛋白的磷酸化

8、,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 indic

9、ates overlapping red and green signals).,雙色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,Ody

10、ssey雙色檢測(cè)的優(yōu)越性,雙色輸出，兩個(gè)目標(biāo)基因同時(shí)檢測(cè)，減少了工作量，而且雜交條件均等，便于兩個(gè)數(shù)據(jù)的對(duì)比 雜交結(jié)果對(duì)比輸出，結(jié)論更加直觀，一目了然 可進(jìn)行準(zhǔn)確的量化分析,In-Cell Western Assay,The Odyssey In-Cell Western (ICW) Assay is a high-throughput approach to simultaneously detect and quantify two separate proteins directly within cells.,In-Cell Western Assay,細(xì)胞培養(yǎng) Culture cel

11、ls to confluency in 96-well or 384-well plates,處理細(xì)胞 Treat cells (inhibitor, stimulator, etc),固定細(xì)胞 Fix cells (3.7%formaldehyde, methanol or Prefer),透化細(xì)胞 Permeabilize cells (0.1%Triton-X 100),Odyssey系統(tǒng)掃描 Scan plate directly and analyze on the LI-COR Infrared Imaging Systems.,In-Cell Western 工作流程,Membr

12、ane Westerns vs ICW,EGF對(duì)ERK磷酸化水平的調(diào)控,700 nm (Total ERK),800 nm (Phospho-ERK),Composite Image Overlay,EGF Concentration,A431 cells stimulated with serial dilutions of EGF to optimize activation of ERK1/2. Phospho-ERK signal was normalized using total ERK signal.,EGF抑制劑對(duì)ERK磷酸化水平的影響,Quantitative and sim

13、ultaneous measurements of total ERK and phosphorylation of ERK in response to EGF in the presence of EGFR inhibitor.,In-Cell Western 軟件模塊,在用客戶提供的結(jié)果,圖片由上海中醫(yī)藥大學(xué)復(fù)雜系統(tǒng)研究中心Dr Liu提供。,Odyssey ICW 的優(yōu)點(diǎn),高速 與普通膜雜交相比，通量提高30倍 ICW 軟件提供便捷的計(jì)算和分析 準(zhǔn)確 紅外檢測(cè)提供最準(zhǔn)確的量化結(jié)果。 避免在細(xì)胞裂解等步驟中蛋白的損失。 操作簡(jiǎn)單 操作步驟簡(jiǎn)單,ICW相關(guān)試劑,目前可以提供4種試劑盒,其他

14、經(jīng)ICW驗(yàn)證的抗體以及kit,應(yīng)用文獻(xiàn)一,P,P,P,P,EGFR,Ras,Raf,Mek,ERK,JAK,STAT1,STAT3,Grb2,SOS,EGF,EGFR Signaling,RAS/RAF/Mek/ERK信號(hào)通路,KSR1,左圖：The effect of KSR1 phosphorylation on MEK1/2 and RSK1 activation.,上圖：Mutation of KSR1 phosphorylation sites enhances the duration of ERK activation.,應(yīng)用文獻(xiàn)二,蛋白質(zhì)芯片掃描,CST PathScan RT

15、K Signaling antibody array kit(7949) kit包括：所有的輔助產(chǎn)品和試劑 R&D 4種適合Licor檢測(cè)試劑盒,小動(dòng)物活體成像-雙色,小動(dòng)物活體成像-附件,軟件功能,儀器軟件 Administration Diagnostics 應(yīng)用軟件 圖象控制 背景扣除 條帶發(fā)現(xiàn)和定位 定量 ICW % 結(jié)果計(jì)算(including normalization) 小動(dòng)物活體分析,Odyssey系統(tǒng)發(fā)表文獻(xiàn)統(tǒng)計(jì),Paper List,Nagashima, K., et al. Genetic and pharmacological inhibition of PDK1 in

16、 cancer cells characterization of a selective allosteric kinase inhibitor J. Biol. Chem 286:6433 (2011) Sneeringer, C.J., et al. Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas PNAS 107:20980

17、 (2010) Cittelly, D. M., et al. Oncogenic HER2D16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors Carcinogenesis 31:2049 (2010) Pan, Y. et al. Sprouty2-modulated Kras signaling rescues Shp2 deficiency during lens and lacrimal glad development. Development

18、 137, 1085-1093 (2010) Markovic, D et al. Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades. Mol Endocrinol. 22(3): 689-706 (2008),總 結(jié),準(zhǔn)確定量提供更完善的數(shù)據(jù)，提升文章檔次 成像效果好背景低，條帶清晰，沒(méi)有