生物學(xué)做Westernblot儀器

1、會(huì)計(jì)學(xué)1生物學(xué)做生物學(xué)做Westernblot儀器儀器解決方案：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ECLOdyssey1跑膠跑膠2轉(zhuǎn)膜轉(zhuǎn)膜3封閉封閉4結(jié)合一抗、洗滌結(jié)合一抗、洗滌5結(jié)合酶標(biāo)記二抗、洗滌結(jié)合紅外染料標(biāo)記二抗、洗滌，成像6發(fā)光試劑盒7暗房曝光成像Antigen on membranePrimary antibody binds to antigenIRDye labeled secondary antibody binds to primary antibody INFRAREDLASER

2、 DIODEINFRARED APD DETECTORON-SCREEN Display信號(hào)持久信號(hào)持久 不需要底物不需要底物 不需要底片不需要底片/暗室暗室TIMETIMECHEMILUMINESCENCE 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 / signalMedium concentration / signalLow concentration / signalSaturationBackground0,00,0INTENSITY紅外熒光 VS 化學(xué)發(fā)光定量線性

3、范圍定量線性范圍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）ECLODYSSEY 倍比稀釋的倍比稀釋的 IRDye 800-標(biāo)記的抗體標(biāo)記的抗體. R2 從從1.5 ng to 0.8 pg 是是 0.99996, 極佳的線性相關(guān)性。極佳的線性相關(guān)性。1.5 ng0.8 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. Sensitiv

4、ity 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).紅外熒光紅外熒光 VS 化學(xué)發(fā)光化學(xué)發(fā)光紅外熒光 VS 化學(xué)發(fā)光成像效果成像效果Odyssey曝光秒曝光秒曝光分鐘曝光分鐘曝光分鐘曝光分鐘曝光分鐘曝光分鐘實(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

5、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 S4Marker S1 S2 S3 S4Odyssey結(jié)果 ECL結(jié)果 信號(hào)強(qiáng)度 51 20 10 4信號(hào)強(qiáng)度 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 Marker10:4:2:1 稀釋 紅外技術(shù)優(yōu)點(diǎn)紅外技術(shù)優(yōu)點(diǎn)低背景低背景NIR DyesBiomoleculesPorphyrinsVisible Fluorophore

6、sPAHs1000 nm800600400200All othersequencers400-650 nmLI-COR700 - 800 nm紅外熒光 vs 可見(jiàn)熒光膜背景信號(hào)膜背景信號(hào)紅外熒光優(yōu)點(diǎn)800 nm700 nmOverlay兩套獨(dú)立激發(fā)檢測(cè)系統(tǒng)700通道：680nm激發(fā) 720nm檢測(cè)800通道：780nm激發(fā) 820nm檢測(cè)No need to strip!No need to re-probe!Target proteinAnti-rabbit 800 channelNormalizer targetAnti-mouse 700 channel* Two-color detec

7、tion requires primary antibodies from different hostsAnti-EGFR and anti-phospho-EGFR antibody specificity in A431 cellsSingle 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 t

8、he red bands above the yellow bands. (yellow indicates overlapping red and green signals).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 Overlay700 nm Channel800 nm ChannelDNA-Protein ComplexUnbound DN

9、A IRDye 800Unbound DNA IRDye 700細(xì)胞培養(yǎng) Culture cells 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)Incubate with IRDye secondary antibodies(e.g.: IRDye 680 and IRDye

10、 800CW)Incubate with 2 primary antibodiesIncubate with 1 primary antibodyIncubate with IRDye secondary antibody anda DNA stain (e.g.: To-Pro-3)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700 nm (Total ERK) 800 nm (Phospho-ERK)Composite Image

11、 OverlayEGF Concentration-02004006008001000120014001600180000.20.40.81.63.126.2512.52550100% Induction of ERKPhosphorylationConcentration EGF (ng/ml)A431 cells stimulated with serial dilutions of EGF to optimize activation of ERK1/2. Phospho-ERK signal was normalized using total ERK signal.Quantitat

12、ive and simultaneous measurements of total ERK and phosphorylation of ERK in response to EGF in the presence of EGFR inhibitor. m nmInhibitor - - 3 1.5 750 375 180 90 45 22 11 5.5EGF - + + + + + + + + + + +700/800 nm 800 nm (Total ERK) 700 nm phosphorylated ERK 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。 目前可以提供4種試劑

13、盒PPP PEGFRRasRafMekERKJAKSTAT1STAT3Grb2SOSEGFEGFR SignalingKSR1 TIMETIMECHEMILUMINESCENCE 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 / signalMedium concentration / signalLow concentration / signalSaturationBackground0,00,0INTENSITY實(shí)驗(yàn)步驟Odyssey:膜+一抗孵育+熒光標(biāo)記抗體孵育+Ody

14、ssey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)狀況紅外技術(shù)優(yōu)點(diǎn)紅外技術(shù)優(yōu)點(diǎn)低背景低背景NIR DyesBiomoleculesPorphyrinsVisible FluorophoresPAHs1000 nm800600400200All othersequencers400-650 nmLI-COR700 - 800 nm紅外熒光優(yōu)點(diǎn)細(xì)胞培養(yǎng) Culture cells 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)Incubate with IRDye secondary antibodies(e.g.: IRDye 680 and IRDye 800CW)Incubate with 2 primary antibodiesIncubate with 1 primary antibodyI