ODYSSEY Classic Infrared Imaging System 教育訓練 吳美萱 騰達行儀器部產品經理
ODYSSEY Classic Infrared Imaging System
ODYSSEY Classic Infrared Imaging System SPECIFICATIONS Image Field Size 25 cm x 25 cm Dynamic Range Manual scan: 4 logs Laser Lifetime 40,000 working hours Resolution 21 337 μm
LI-COR Detection System 680 & 780 nm solid state laser diodes (A) No spectral overlap Low power consumption Long life lasers (>40,000 hours) Silicon avalanche photodiode detectors (C) Resolution 21-337 microns Internal hard drive Secure network connectivity B C D A A C
Chemiluminescent Western Detection FILM / CCD light Substrate Antigen Primary Antibody Secondary antibody http://www.licor.com/bio/applications/chemiluminescent_western_blot/
Infrared Western Detection Infrared APD Detector Infrared Laser Diode Image Studio Software Antigen Primary Antibody Secondary antibody http://www.licor.com/bio/applications/quantitative_western_blots/
Limitations of Chemiluminescence Enzymatic reaction Signal not proportional to antigen Multiple targets require stripping
Enzymatic vs. Infrared Chemistry Chemiluminescence Not directly proportional NIR Fluorescence Directly proportional Detector Saturation 2 : 1 3 : 1 1 : 2 2 : 1 2 : 1 2 : 1 Signal Background 0 5 10 15 HRP Enzyme Reaction Minutes 0 6 12 18 Fluorescent Immuno-complex Months Protein Concentration
Fluorescent Spectra
Excellent SNR Odyssey (Near Infrared) ECL Plex (visible fluorescence) No EGF + EGF No EGF + EGF 800nm (ERK) 633nm (ERK) Cy5 700 nm (perk) 532nm (perk) Cy3 2-fold serial dilutions of A431 cell lysate
Multiplexed Quantitative Assay 700 nm Channel Image 800 nm Channel Image Overlaid Images Simultaneously collects digital images of the signals from each fluorophore Images can be viewed and quantified individually or as an overlaid image
NIR Fluorescence
Odyssey Family of Instruments APPLICATIONS
Infrared Western Blots
Multiplex Western blot approaches. Two different targets can be detected A phospho-antibody can be combined with an antibody against the unmodified target protein (pan-ab) for multiplex phosphorylation analysis
Infrared Western Blots
Infrared Western Blots
Phosphorylation Detection JNK Phosphorylation in response to LPS treatment Activation of MAPK by Corticotropin Releasing Hormone Biol. Proced. Online 2008; 10(1):20-28 Mol. Endocrinol. 20:3179-3195 (2006)
MicroWestern Array Jones laboratory Ben May Institute for Cancer Res. Univ. of Chicago Ciaccio et al. Nature Methods 7:148-155 (2010)
MicroWestern Array Ciaccio et al. Nature Methods 7:148-155 (2010)
In-Cell western Assay
In-Cell Western Assay Immunofluorescent Approach ICW: A high-throughput approach to simultaneously detect and quantify two separate proteins directly within cells.
Stimulation of ERK Phosphorylation in A431 Cells by Treatment with EGF 1 o Ab - + + + + + + + + + + + EGF - - 0.2 0.4 0.8 1.6 3.12 6.25 12.5 25 50 100 ng/ml Replicates Overlaid Images 700 nm (Total ERK) 800 nm (Phospho-ERK) 700: Alexa Fluor 680 800: dye candidate equivalent to IRDye TM 800CW Chen, H., Kovar, J., Sissons, S., Cox, K., Matter, W., Chadwell, F., et al. (2005). A Cell Based Immunocytochemical Assay For Monitoring Kinase Signaling Pathways And Drug Efficacy. Analytical Biochemistry, 136-142.
Stimulation of ERK Phosphorylation in A431 Cells by Treatment with EGF 1800 1600 1400 ERK phosphorylation 1200 1000 800 600 400 200 0 0 0.2 0.4 0.8 1.6 3.12 6.25 12.5 25 50 100 EGF ng/ml Dose-dependent increase in ERK phosphorylation
Protein Phosphorylation: Timing/Kinetics of Signal Transduction Kinetics of cellular β-catenin accumulation upon stimulation with Wnt3a A A) Time- and dose-dependent accumulation of cellular β-catenin. B) Quantification of β-catenin accumulation. Graph shows two independent experiments, each done in quadruplicate. B Hannoush RN (2008) Kinetics of Wnt-Driven β-catenin Stabilization Revealed by Quantitative and Temporal Imaging. PLoS ONE 3(10): e3498. doi:10.1371/journal.pone.0003498 http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003498
Receptor Localization In-Cell Western On-Cell Western
On-Cell Western Assay Optical Agent Binding Assay Optical Agent Competitive Challenge
Protein Array
Protein Arrays Antibody arrays challenged with IR-labeled protein extracts Ab Array Incubate Wash Scan IR label protein extract IR - protein Spot intensity correlates with expression levels. Ivanov, S. S., Chung, A. S., Yuan, Z.-l., Guan, Y.-j., Sachs, K. V., Reichner, J. S., et al. (2004). Antibodies Immobilized as Arrays to Profile Protein Post-translational Modifications in Mammalian Cells. Molecular & Cellular Proteomics, 788-795.
Reverse Phase Protein Arrays Selected cells are laser captured and removed from the tissue Protein lysates are arrayed into nitrocellulose slides Extraction buffer is applied to laser-captured cells 350 μm 350 μm Slides are probed with antibody of choice, then developed by chemiluminescence, fluorescence, or colorimetric means
Reverse Phase Protein Arrays Breast Cancer Biopsies Jurkat Cells Total ERK Phospho-ERK Pierobon, M., Belluco, C., Liotta, L. A., & Petricoin III, E. F. (2011). Reverse Phase Protein Microarrays for Clinical Applications. In Methods in Molecular Biology (pp. 3-12). Springer Protocols.
Tissue section imaging
Tissue Sections Sagittal section of mouse brain immunostained for CB1 and D2 receptors. The left images are pseudocolor overlays with CB1 receptor in red and D2 receptor in green. Kearn, C. S. (2004). LI-COR Application Note
Tissue Sections High Throughput Imaging of Brain Sections Kearn, C. S. (2004). LI-COR Application Note
ELISA
ELISA, FLISA or Infrared ELISA? FLISA ELISA IR ELISA IRDye Streptavidin Biotinylated or IRDye Secondary Antibody X X X X X X X X X X
ELISA (AP and HRP 680 Substrates) Same sensitivity Longer dynamic range (no colorimetric limit)
Other Applications
EMSA / Gel Shift Assays Chia, D. J., Ono, M., Woelfle, J., Schlesinger-Massart, M., Jiang, H., & Rotwein, P. (2006).JBC, 3190-3197.
1-D / 2-D Gel Documentation Coomassie gel documentation (700 nm) Coomassie stained 2-D gel documentation (700 nm)
Applications for LI-COR Products http://www.licor.com/bio/applications/
LI-COR Applications REAGENTS & CONSUMABLES
LI-COR Reagents and Consumables https://www.licor.com/bio/products/reagents/index.html
Reagent Flexibility Compatible with many dyes and stains 790 800 +++ DyLight 700 +++ DyLight 800 +++ 800
LI-COR Applications Application Protocols TIPS FOR WESTERN BLOTTING
Western Blotting - Direct Fluorescent Detection Infrared dye (IRDye 680RD, IRDye 800CW) Antigen on Membrane Primary antibody binds to antigen IR-labeled secondary antibody binds to primary antibody SCAN ON ODYSSEY Time not critical (days, weeks, months). No substrates required. No film / darkroom required.
Odyssey Western Protocol Protein Gel Electrophoresis Electro-transfer on membrane (low background PVDF, NC) Blocking (Try Odyssey Blocking buffer) Tween-20 SDS Hyb. With Primary Antibody As usual Hyb. With Infrared (IR)-labeled Secondary Antibody 1:15,000 ( 1:5000 ~ 1:25,000) Scanning with Odyssey
Factors That Alter the Performance of a Western Blot A low background membrane is essential for fluorescent WB success. A. PVDF Membrane: Millipore Immobilon FL 700 nm Channel Scan Intensity = 7 800 nm Channel Scan Intensity = 8 Millipore Immobilon P BioRad Immun-Blot Pall BioTrace PVDF Perkin Elmer PolyScreen Amersham Hybond -P
Factors That Alter the Performance of a Western Blot Antibody performance can sometimes be compromised by the blocker chosen. B. Blocking Buffer: Western blots detected with anti- PKCα and IRDye 800CW Goat antimouse. pakt β-tubulin Odyssey Blocker I-Block 5% BSA T293 Cells Stimulated with TGF- at 0, 2.5, and 5 min
Factors That Alter the Performance of a Western Blot C. Miscellaneous Contamination: Good Westerns Gone Bad
Imaging Factors That Can Affect Western Blot Results Handling of the blot Air bubbles affect image appearance. Scanning of wet and dry Western blots. 對比度 對比度
Scanning of wet and dry Western blots. wet dry
Scanning Procedures
Getting Started Odyssey 系統開機按壓主機電源開關按鈕, 待面板顯示即開機完成 連接軟體 1. 開啟電腦桌面上軟體 2. 點選檔案存取路徑檔案夾, 按下 OK 3. 輸入使用者代號與密碼 3. 確認 Status 顯示 Ready
Western blot/gel 擺放 Membrane / gel 正面朝下 Scan Boundary
Microtiter plate 擺放 Plate 正面朝上, 緊靠 Alignment Guide 放置
掃描條件設定 點 選擇預設之掃瞄條件或 / 及 設定各參數 點選 手動調整掃瞄訊號強度 以滑鼠拖曳出實際欲掃瞄的區域 根據 membrane 在掃瞄玻璃面板上的刻度 若選擇掃瞄完整 plate 則不需更改 點 即開始掃瞄
掃描完成
掃描完成
Post-Scanning 掃描影像檢視 影像檔案列表
Post-Scanning 掃描影像輸出 /Export -> 預設輸出影像檔案的格式為 150 dpi PNG 預設輸出影像檔案的格式為 600 dpi TIFF 輸出原始檔案以利到它臺電腦上供 Image Studio 軟體再進一步分析
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