產品專員李宗樹奇異亞洲醫療設備股份有限公司. Typhoon Overview

Transcription

1 Typhoon - A Versatile Imaging System 產品專員李宗樹奇異亞洲醫療設備股份有限公司 1/ Typhoon Overview Applications - Radioisotopic (Macroarrays) - Chemiluminescence (Westerns) - Fluorescence (Protein, DNA, Multi-Color) Technical Summary Advantages 2/ 1

2 Typhoon Applications by label/detection Radioactive Non-radioactive Colorimetric Chemiluminescent Fluorescent 3/ Typhoon Applications 1D gel/blot by image analysis 2D Differential display Biotech GFP Microplate analysis Microarray 4/ 2

3 Typhoon storage phosphor imaging Storage phosphor detection of radioactivity > 3 H, 125 I, 14 C, 35 S, 33 P, 32 P, 18 F, 99m Tc, etc. The proven storage phosphor capabilities > Limit of Detection (LOD): 2 dpm/mm 2 for 14 C > Linear Range: 5 orders with <7.5% std. dev. > Uniformity: + 5% over entire scan area 5/ Storage Phosphor - How it works BaFBr: Eu +2 phosphor at ground state BaFBr - : Eu +3 phosphor absorbs x-rays, a, b or g BaFBr: Eu +2 * excited F-centre absorbs red laser light to enter unstable state BaFBr: Eu +2 phosphor returns to ground state Emitting Europium luminescence 6/ 3

4 Different from film exposure Film - Not quantitative - Only one copy of the data, not digital - Low dynamic range Screen - Quantitative - Save time: 1/5 to 1/10 exposure than film - High dynamic range Use Image Eraser to erase Image DO NOT use detergent on it 7/ Chemiluminescence Direct imaging and quantification without exposures to films or screens Chemiluminescence - Westerns ECL ECL Plus SuperSignal West Femto SuperSignal West Dura CDPstar 8/ 4

5 Typhoon Applications ECL Plus westerns Typhoon chemiluminescence mode high sensitivity Hyperfilm ECL (PDSI) 1 minute exposure Typhoon Applications Fluorescent Westerns Enzyme-amplified >ECF (Enhanced Chemifluorescence) >DDAO Phosphate Direct >Cy3 >Cy5 5

6 General WB Gel separation ECL Plex Gel separation Gel transferring Gel transferring Blocking Blocking 1st Ab binding 1st Ab binding 2nd HRP Ab binding 2nd ECL Plex Ab binding ECL develop *Fluorescent Scan Film or CCD detect 11 / Western Applications ECF 532/526SP Tubulin detected in rat brain homogenate DDAO Phosphate 633/670BP30 6

7 Western Applications Cy3 532/580BP30 Tubulin detected in rat brain homogenate Cy5 633/670BP30 Western Applications Cy3 & Cy5 Dual Target Western Cy3 (green) Cy5 (red) - actin - tubulin 7

8 Typhoon Applications DNA Gel Stains Ethidium bromide Vistra Green SYBR Green I SYBR Gold DNA Gel Stain Applications EtBr (532/610DF30) DNA Mass Ladder SYBR Green I (532/526SP) 8

9 DNA Gel Stain Applications SYBR Green I agarose 384(96x4)PCRs/scan front end to high throughput capillary sequencing Courtesy ART - Sunnyvale 17 / Differential Display Analysis lung liver lung liver lung liver Rat Liver and Lung cdna labeled Cy5 and electrophoresed on a 6% denaturing polyacrylamide gel. 9

10 DNA Gel Stain Applications PicoGreen 532/526SP Lambda DNA PicoGreen - DNA Solution Quantitation LOD = 10 ng/ml Fluorescence Linearity = 200-fold 400 R 2 = DNA (ng/ml) Typhoon Applications Protein Gel Stains SYPRO Orange SYPRO Red SYPRO Ruby (2-D gels) DeepPurple 10

11 Protein Stain Applications SYPRO Red 532/610BP30 SYPRO Red Linearity BSA Protein Fluorescence 4.E+06 4.E+06 3.E+06 3.E+06 2.E+06 2.E+06 1.E+06 5.E+05 0.E+00 R 2 = [BSA] (ng per band) Protein Stain Applications SYPRO Ruby 1-D SYPRO Ruby2-D 532/610BP30 Protein Mass Ladder Carrot Seed Protein 11

12 Typhoon Applications Fluorescent Covalent DNA Labels Polyacrylamide Gels Fluorescein/FAM HEX, TAMRA, ROX Cy3, Cy5 Covalent DNA Labels Applications Mapping Agarose gel +3 mm plane Genomic mapping 3-color: >FAM (blue) >HEX (green) >ROX (red) (Courtesy Laurie Gordon, LLNL, Livermore, CA, USA) 12

13 Gel Shift Assay Mnt repressor protein 180bp end-labeled operator DNA fragment Sandwich gel 4-color: FAM (green), HEX (red), TAMRA (blue) and ROX (yellow) FluorSep processed (Courtesy Chris Man, WashU, St Louis, MO, USA) Microarray Corning slide spotted with cdna from APB ScoreCard control and cloned cdna from Incyte Genomics Hybridized with 25pmoles of Cy3 and Cy5 labelled mrna: Liver - Cy5 Skeletal Muscle - Cy3 26 / 13

14 27 / Challenges for 2D electrophoresis n n n n n Sensitivity (CBB) Linear Dynamic Range (CBB Silver) Reproducibility (Gel-to-gel variation) Accuracy (Statistics needed) Consumption (Gels, time, samples) 14

15 Presentation of the problem 6 gels made from the same sample, run in parallel (SYPRO Ruby) Bad guy Nice guy 29 / GE Healthcare Jörgen Östling, AstraZeneca R&D Mölndal, Sweden. June 2004 Advantage of fluorescent detection: increased dynamic range 2-D DIGE 3-D plots of same spot areas on gel Silver staining 30 / GE Healthcare 15

16 Measuring induced biological change Differences due to disease state, drug treatment, life cycle stage Experimental factors Differences in IEF/SDS-PAGE conditions Gel distortions user-to-user variation Data analysis user-specific editing and interpretation Intrinsic differences that occur within populations eg animal-to-animal, plant-to-plant or culture-toculture, subjected to identical conditions Induced biological change - what we want to measure System variation (gel-to-gel variation) Inherent biological variation 16

17 Difference gel electrophoresis (DIGE) 2-D DIGE using an internal standard Cy2 Cy3 Cy5 Standard Sample 1 Sample 2 Internal standard and 2 samples per gel Cy2 Cy3 Cy5 Standard Sample 3 Sample 4 Gel A Gel B Cy2 Cy3 Cy5 Gel C Standard Sample 5 Sample 6 17

18 2-D DIGE: internal standardization Standard (Cy TM 2) Sample 1(Cy3) Sample 2 (Cy5) Without internal standard Gel A With internal standard Standard (Cy2) Sample 3(Cy3) Sample 4 (Cy5) Gel B Virtual elimination of gel variation: induced biological change with statistical accuracy capable of revealing differences in abundance of less than 10% between samples Normalisation of spots between gels Not normalised to standard Normalised to standard 1.79-fold with greater than 99.9% t-test confidence. 18

19 1 color 2D vs. DIGE -Number of gels 2-D DIGE/internal std/decyder TM 1 colour 2-D 8 samples 2 samples per gel = 4 gels x 3 biological replicates 0 gel replicates = 12 gels total = 6 runs by SE600 or 2 runs by DALTsix = 6 days (3 days/run) 8 samples 1 sample per gel = 8 gels x 3 biological replicates x 3 gel replicates = 72 gels total = 36 runs by SE600 or 12 runs by DALTsix = 36 days (3 days/run) Benefits of Ettan DIGE Reduce Reduce Reduce Ensure number of gels hands on time amount of sample required accurate quantification of differences in protein abundance through the use of an internal standard Increase the number of real hits *) *) Friedman et al. Proteomics 2004; 4(3):

20 DIGE paper reference Typhoon Overview Applications > Radioisotopic (Macroarrays) > Chemiluminescence (Westerns) > Fluorescence (Protein, DNA, Multi-Color) Technical Summary Advantages 40 / 20

21 Typhoon Versatility Sample Requirement: Fluorescence (633nm, 532nm, 488nm) Radioactive Chemiluminescence samples Gels and sandwich gels Blotting membranes Macroarray and microarray chips Micro-plates Tissue sections (not in situ) Radio-TLC & X-Ray diffraction Large format: 43 x 35 cm maximum Scans mounted & un-mounted screens Most screens even from other suppliers 41 / Typhoon - Sensitivity Optical Design: Confocal optics 25, 50, 100, 200, 500, 1000 µm pixels 5 log dynamic range Twin PMT s, one is cooled Objective changer 0 & +3mm Emission and excitation: 6 std emission filters, total 13 filter seats 3 std beam splitters 560, 580nm & 630nm 2 excitations 532nm (green laser) & 633nm (red laser) Blue laser is optional 42 / 21

22 Typhoon excitation & collection Glass Platen Gel Scan Head Lens Objective Lens Changer Assembly Lens 1 Lens 2 Laser Line Filter PMT & Filter Changer Assembly Mirrors Shutter Assembly 532nm 633nm Confocal optics Dual excitation Dual emission Focusing Optics Collimating Lens B/S B/S B/S Emission Filters Focusing Lens Focusing Lens PMT 1 PMT 2 43 / Sample Glass platen Objective Excitation Laser Z-Plane Mirror Laser reflection filter Collection Lens Advantages of Confocal Optics Rejection of Scatter Flat Field Focus Control Fiber Optic Cable 44 / 22

23 Galvonometer Sample Tray f-theta Lens Laser Galvo Mirror 45 / Galvanometer scanning - Distortion problem Galvanometer Two problems 1. Very obvious flaring of the high activity samples 2. Distortion of wells Typhoon 46 / 23

24 Typhoon free gel Typhoon Scan Setup Focal Depth > Platen setting default for storage phosphor choice for membranes, thin gels on platen > + 3mm (3 +/- 0.5mm above platen surface) thick agarose gels microplates polyacrylamide sandwich gels (glass plate) 3mm platen (support) gel Emission filters Filter Fluorophore 520DF30 ECL Plus, Cy2 526SP Fluorescein, FAM, SYBR, ECF, PicoGreen 555DF20 TET, HEX, R-6G 560LP Multiple fluors (single channel scans) 580DF30 Cy3, HEX, TAMRA, SYPRO Orange 610DF30 ROX, C-X-R, SYPRO Red/Ruby, EtBr 670DF30 Cy5 Emission filter assembly for custom filters is available * 520DF30 is optional filer 48 / 24

25 Technical Manual: Fluorescence Imaging 49 / Excitation & Emission Typhoon 580BP30 575LP Filter Emission Filter: - The more is the better - More specific is more better 50 / 25

26 51 / SYPRO Ruby ng Excitation: 457 nm Emission: 610BP30 Excitation: 488 nm Emission: 610BP30 Excitation: 532 nm Emission: 610BP30 S/N > 3 Pixel Size: 200µm; PMT: 550V; Focal Depth: Platen All three laser lines offer same sensitivity for SYPRO Ruby- stained 1D protein gel 52 / 26

27 Thanks for your attention 53 / 27