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1 ODYSSEY Classic Infrared Imaging System 教育訓練吳美萱騰達行儀器部產品經理

2 ODYSSEY Classic Infrared Imaging System

3 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 μm

hours) Silicon avalanche photodiode detectors (C) Resolution 21-337

4 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 microns Internal hard drive Secure network connectivity B C D A A C

5 Chemiluminescent Western Detection FILM / CCD light Substrate Antigen Primary Antibody Secondary antibody

6 Infrared Western Detection Infrared APD Detector Infrared Laser Diode Image Studio Software Antigen Primary Antibody Secondary antibody

7 Limitations of Chemiluminescence Enzymatic reaction Signal not proportional to antigen Multiple targets require stripping

8 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 HRP Enzyme Reaction Minutes Fluorescent Immuno-complex Months Protein Concentration

9 Fluorescent Spectra

10 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

digital images of the signals from each fluorophore Images

11 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

12 NIR Fluorescence

13 Odyssey Family of Instruments APPLICATIONS

14 Infrared Western Blots

15 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

16 Infrared Western Blots

17 Infrared Western Blots

18 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: (2006)

19 MicroWestern Array Jones laboratory Ben May Institute for Cancer Res. Univ. of Chicago Ciaccio et al. Nature Methods 7: (2010)

20 MicroWestern Array Ciaccio et al. Nature Methods 7: (2010)

21 In-Cell western Assay

22 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.

5 25 50 100 ng/ml Replicates Overlaid Images 700 nm (Total ERK) 800 nm (Phospho-ERK) 700: Alexa Fluor 680 800: dye candidate

23 Stimulation of ERK Phosphorylation in A431 Cells by Treatment with EGF 1 o Ab EGF ng/ml Replicates Overlaid Images 700 nm (Total ERK) 800 nm (Phospho-ERK) 700: Alexa Fluor : 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,

24 Stimulation of ERK Phosphorylation in A431 Cells by Treatment with EGF ERK phosphorylation 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

25 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: /journal.pone

26 Receptor Localization In-Cell Western On-Cell Western

27 On-Cell Western Assay Optical Agent Binding Assay Optical Agent Competitive Challenge

28 Protein Array

29 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,

30 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

Reverse Phase Protein Microarrays for Clinical

31 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.

32 Tissue section imaging

33 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

34 Tissue Sections High Throughput Imaging of Brain Sections Kearn, C. S. (2004). LI-COR Application Note

35 ELISA

36 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

37 ELISA (AP and HRP 680 Substrates) Same sensitivity Longer dynamic range (no colorimetric limit)

38 Other Applications

39 EMSA / Gel Shift Assays Chia, D. J., Ono, M., Woelfle, J., Schlesinger-Massart, M., Jiang, H., & Rotwein, P. (2006).JBC,

40 1-D / 2-D Gel Documentation Coomassie gel documentation (700 nm) Coomassie stained 2-D gel documentation (700 nm)

41 Applications for LI-COR Products

42 LI-COR Applications REAGENTS & CONSUMABLES

43 LI-COR Reagents and Consumables

44 Reagent Flexibility Compatible with many dyes and stains DyLight DyLight

45 LI-COR Applications Application Protocols TIPS FOR WESTERN BLOTTING

IR-labeled secondary antibody binds to primary antibody SCAN ON ODYSSEY Time

46 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.

47 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

PVDF Membrane: Millipore Immobilon FL 700 nm Channel Scan Intensity = 7 800 nm

48 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 = nm Channel Scan Intensity = 8 Millipore Immobilon P BioRad Immun-Blot Pall BioTrace PVDF Perkin Elmer PolyScreen Amersham Hybond -P

49 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

50 Factors That Alter the Performance of a Western Blot C. Miscellaneous Contamination: Good Westerns Gone Bad

51 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

52 Scanning of wet and dry Western blots. wet dry

53 Scanning Procedures

54 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

55 Western blot/gel 擺放 Membrane / gel 正面朝下 Scan Boundary

56 Microtiter plate 擺放 Plate 正面朝上, 緊靠 Alignment Guide 放置

57 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

58 掃描完成

59 掃描完成

60 Post-Scanning 掃描影像檢視影像檔案列表

61 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

62 We can help you get high-quality data!

LI-COR IR Detection

LI-COR IR Detection 吳美萱儀器部產品經理 The Path to Quantitative Analysis Quantitative Westerns Solution Provider Standard in the Industry Over 20,000 Users More than 2,900 Publications Application Flexibility Optimized Reagent Solutions