LI-COR IR Detection

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1 吳美萱儀器部產品經理 The Path to Quantitative Analysis

2 Quantitative Westerns Solution Provider Standard in the Industry Over 20,000 Users More than 2,900 Publications Application Flexibility Optimized Reagent Solutions Excellent Technical Support Easy Transition Protocols

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

3 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 B Internal hard drive Secure network connectivity A A C C D

4 Why Use Infrared Fluorescence? Many natural and synthetic compounds autofluoresce in the visible region Nitrocellulose, PVDF, nylon, cellular content Low background fluorescence in IR range translates to excellent sensitivity

5 Applications

6 Odyssey Application Flexibility Western Detection Two-Color Blots In-Gel Westerns Cell-Based Assays In-Cell Western TM Assay On-Cell Western TM Assay RNAi Microwell Assays FLISA Transcription Factor Arrays Protein Gel Documentation 1D/2D Coomassie Nucleic Acids EMSA/Gel Shift DNA Staining Small Animal Imaging Organ + Tissue Section Imaging

7 Easy Transition From Chemiluminescent Perform electrophoresis and transfer as usual Block in Blocking buffer 30 minutes to an hour Dilute PRIMARY Antibody and incubate as usual Wash Dilute IRDye labeled SECONDARY Antibody and incubate 1 hour Wash Blot is ready for imaging * Easy transition from ECL *

8 Direct Infrared Detection ON-SCREEN Display INFRARED APD DETECTOR INFRARED LASER DIODE Antigen on membrane Primary antibody binds to antigen IRDye labeled secondary antibody binds to primary antibody

Direct Infrared Detection Quantative Western Tf protein: Dilutions of transferrin blotted on nitrocellulose, detected with rabbit anti-tf primary and IRDye secondary antibody 1.

9 Direct Infrared Detection Quantative Western Tf protein: Dilutions of transferrin blotted on nitrocellulose, detected with rabbit anti-tf primary and IRDye secondary antibody 1. No exposure/integration time No darkroom 2. No enzyme kinetics Not time dependent; no substrates 3. Large linear range of chemistry 4. No signal diffusion Band clarity 5. Sensitivity equivalent or better than chemiluminescense

Quantification Accuracy - Linearity Linear range is the range over which bands can be accurately quantified, in contrast to dynamic range which is the total range of the detection hardware. 3.0 ng 0.

10 Quantification Accuracy - Linearity Linear range is the range over which bands can be accurately quantified, in contrast to dynamic range which is the total range of the detection hardware. 3.0 ng 0.37 pg Protein Concentration R 2 = Intensity >4 logs linear range Two-fold serial dilutions of antibody (6 ng to 0.19 ng) were spotted on nitrocellulose (above). R 2 from 3.0 ng to 0.37 pg was 0.998, demonstrating excellent linearity even at low concentration.

Linearity Data Odyssey Direct Infrared Detection Integrated Intensity 50000 40000 30000 20000 R 2 = 0.

11 Linearity Data Odyssey Direct Infrared Detection Integrated Intensity R 2 = Antigen ng 0.3 pg-0.6 ng detection limit (all 15 protein spots) Signal IS proportional to amount of antigen

12 Two-Color Simultaneous Detection This shows phospho-egf (Red, 700 Channel) normalized to Actin (Green, 800 Channel) in A-431 cells treated with a dose response of EGF. * Two-color detection requires primary antibodies from different hosts

13 Monitoring EGF Receptor Phosphorylaytion Anti-EGFR and anti-phospho-egfr antibody specificity in A431 cells 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). Single color images (B and C) can be overlaid (A) to show both total protein and phosphorylated protein.

14 In-Cell Western TM Assay Immunofluorescent Approach The Odyssey In-Cell Western (ICW) Assay is a highthroughput approach to simultaneously detect and quantify two separate proteins directly within cells.

In-Cell Western Multiple Target Analysis Experimental Data um nm

5 EGF: - + + + + + + + + + + + Normalized level of EGFR

channel 700 channel Data one target (EGFR) and PD168393; 800 channel

15 In-Cell Western Multiple Target Analysis Experimental Data um nm Inhibitor: EGF: Normalized level of EGFR phosphorylation was used to generate IC-50 curve for PD channel 700 channel Data one target (EGFR) and PD168393; 800 channel = normalization; 700 channel = experimental Analytical Biochemistry, March 2005 Vol. 338, Issue 1, pp

Cells were normalized to TO-PRO-3 staining (red).

16 On-Cell Western TM Assay Analysis of Cell Surface Proteins Optical Agent Binding Assay: A dose response was noted when A431 cells were incubated with increasing concentrations of IRDye 800CW EGF (green). Cells were normalized to TO-PRO-3 staining (red). Optical Agent Competitive Challenge: Increasing concentrations of unlabeled EGF successfully competed for binding sites with IRDye 800CW EGF (green). Cells were normalized to TO-PRO-3 staining (red).

17 Molecular Imaging Tissue Sections Vertebrae cross-section Femurs 21 um Odyssey scans of bone sections IRDye 800CW BoneTag deposition presented in green and autofluorescence in red.

18 Protein Detection In Gel Western 1-D / 2-D Coomassie Nucleic Acid Detection EMSA / Gel Shift Assays DNA Staining Microwell Assays ELISA/FLISA Transcription Factor Arrays Protein Arrays Plated based Membrane based Slide based Quansys Multiplex ELISA (cytokine/chemokines) Biosciences GE Array TM Additional Applications

19 EMSA / Gel Shift Assays IRDye labeled DNA fragments Data based on the interaction of a protein and DNA fragment Shift in mobility is visualized when scanned

EMSA / Gel Shift Assays Characterization of

Molecular Biology, Oregon Health and Science

Sciences, Virginia Polytechnic Institute and

20 EMSA / Gel Shift Assays Characterization of Distinct Stat5b Binding Sites That Mediate Growth Hormone-stimulated IGF-I Gene Transcription Department of Biochemistry and Molecular Biology, Oregon Health and Science University Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University J. Biol. Chem., Vol. 281, Issue 6, , February 10, 2006

21 Odyssey Application Flexibility

22 Solutions for Applications

23 Optimized Reagent Solutions Optimized Kits for Western Blots Optimized Kits for Cell-Based Assays EMSA Buffer Kit Optical Probes for Tumor Imaging Labeling Kits for Antibodies, Proteins Multiple Infrared Dyes Labeled Antibodies and Conjugates

24 Reagent Solutions Detection of other dyes is possible Many different conjugates are available to meet research needs Use may be limited to one channel depending on wavelength of chosen dye

25 IRDye Conjugated Antibodies and Reagents IRDye700DX Conjugates IRDye800 Conjugates IRDye800CW Conjugates

26 Software Solutions Instrument software Administration Diagnostics Application software Imager control Background subtraction Band finding and sizing Quantification ICW % response calculations (including normalization) Small animal imaging analysis

27 Application Protocols

28 Application Protocols Get the help you need, when you need it.

29 Western Blotting - Direct Fluorescent Detection Infrared dye (IRDye680 LT, IRDye800CW) 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.

30 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 1:1000 to 1:5000 Hyb. With Infrared (IR)-labeled Secondary Antibody 1:15,000 ( 1:5000 ~ 1:25,000) Scanning with Odyssey

31 Factors That Alter the Performance of a Western Blot A low background membrane is essential for fluorescent WB success. A. PVDF Membrane: 700 nm Channel 800 nm Channel Millipore Immobilon FL Millipore Immobilon P BioRad Immun-Blot Amersham Hybond -P

32 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 T293 Cells Stimulated with TGF- at 0, 2.5, and 5 min Odyssey Blocker I-Block 5% BSA

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

34 Scanning Procedures

35 File/New 開啟新檔案

36 Western blot/gel 擺放 Membrane / gel 正面朝下, 上方靠近操作者, 長的一邊橫放 Scan Boundary

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

38 掃描條件設定 LI-COR Presets:

39 設定掃描範圍 Click and hold down the mouse button in the lower left corner of the area to be scanned. Drag the cursor to the upper right corner of the area to be scanned and release the mouse button.

40 開始掃描

41 掃描期間影像即時顯示顯示剩餘之時間顯示已完成之百分比

42 掃描完成

43 Post-Scanning 掃描影像檢視 Double click

44 Post-Scanning 掃描影像檢視主工具列影像對比度調整影像顯示大小調整 700/800 單色畫面轉換雙色 / 單色畫面轉換開啟新掃描彩色 / 灰階畫面轉換

45 Post-Scanning 影像對比度調整

46 Post-Scanning 掃描影像輸出 File/Export Image->

47 Thank You! Western Detection Two-Color Blots In-Gel Westerns Cell-Based Assays In-Cell Western TM Assay On-Cell Western TM Assay RNAi Microwell Assays FLISA Transcription Factor Arrays Protein Gel Documentation 1D/2D Coomassie Nucleic Acids EMSA/Gel Shift DNA Staining Small Animal Imaging Organ + Tissue Section Imaging

PowerPoint Presentation

PowerPoint Presentation 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