Typhoon™ FLA 9500 biomolecular imager
Contents
1. Typhoon FLA 9500 1 28 9969 43 Includes 473 nm 532 nm and 635 nm lasers filter tray IP filter LPB filter LPG filter LPR filter LPR filter for ch2 BPB1 filter BPG1 filter Fluor Stage Membrane Weight Phosphor Stage Low Fluorescent Glass Plate Stage Multi Stage with TP plug in Fluorescent plate for digitization capture software USB cable mains cables EU and USA User Manual Getting Started Guide One license of ImageQuant TL software is provided with Typhoon FLA 9500 Upgrades and accessories Quantity Code number BAS IP MS 2040 E 1 28 9564 74 Phosphorimaging plate 20 x 40 cm multipurpose BAS IP MS 2025 E 1 28 9564 75 Phosphorimaging plate 20 x 25cm multipurpose BAS IP MS 3543 E 1 28 9564 76 Phosphorimaging plate 35 x 43 cm multipurpose BAS IP SR 2040 E 1 28 9564 77 Phosphorimaging plate 20 x 40 cm high resolution BAS IP SR 2025 E 1 28 9564 78 Phosphorimaging plate 20 x 25 cm high resolution BAS IP TR 2040 E I 28 9564 81 Phosphorimaging plate 20 x 40 cm for Tritium detection BAS IP TR 2025 E 1 28 9564 82 Phosphorimaging plate 20 x 25 cm for Tritium detection BAS IP ND 2040 E 1 29 0171 33 Phosphorimaging plate 20 x 40 cm for Neutron detection BAS IP ND 2025 E 1 29 0171 39 Phosphorimaging plate 20 x 25 cm for Neutron detection Exposure Cassette 1 63 0035 44 Unmounted Screen 20 x 25 cm Exposure Cassette 1 63 0035 45 Unmounted Screen 35 x 43 cm FLA Image Eraser 1 28 952. in the US and other countries Cy3 UTP or Cy5 UTP Cy3 5 dCTP or Cy5 5 dCTP Cy3 CTP or Cy5 CTP These products are manufactured for GE Healthcare UK Limited by Perkin Elmer Life Sciences under US patent numbers 5047519 and 5151507 The cyanine dyes in the product are manufactured under an exclusive license from Carnegie Mellon University under US patent numbers 5 268 486 and equivalent patents in the US and other countries The purchase of CyDye products includes a limited license to use the CyDye products for internal research and development but not for any commercial purposes A license to use the CyDye products for commercial purposes is subject to a separate license agreement with GE Healthcare Commercial use shall include 1 Sale lease license or other transfer of the material or any material derived or produced from it 2 Sale lease license or other grant of rights to use this material or any material derived or produced from it 3 Use of this material to perform services for a fee for third parties including contract research and drug screening If you require a commercial license to use this material and do not have one return this material unopened to GE Healthcare Bio Sciences AB Bjorkgatan 30 SE 751 84 Uppsala Sweden and any money paid for the material will be refunded 2011 2012 General Electric Company All rights reserved First published Oct 2011 All goods and services are sold subject to the terms and conditio
3. system The detection limit was 25 pg and the linear dynamic range DR was 4 3 orders of magnitude accommodates up to four computer controlled filter positions at any time Custom filters can be easily installed by the user Stages Fig 4 give the correct positioning and stability for optimal imaging of a range of sample types Samples that can be scanned include agarose and polyacrylamide gels membranes DIGE gels radioisotope labeled samples using a phosphorimaging plate as well as microplates and glass slides with the titer plate TP plug in The system can simultaneously scan two DIGE gels each measuring up to 21 5 x 27 5 cm with the Low Fluorescent Glass Plate stage The stages are easily removed from the system for cleaning Typhoon FLA 9500 comes equipped with both bi alkali and multialkali photomultiplier tubes PMT This combination provides excellent detection over a very broad spectrum Each PMT is selected for optimal response to the detected emission wavelength The bialkali PMT 1 is suitable for phosphorimaging and detection of dyes emitting blue and green light e g Cy2 and Cy3 whereas the multialkali PMT 2 is optimal for detection of dyes emitting yellow red e g Cy5 far red and infrared light e g IRDye800 2 29 0044 13 AB Fig 4 A The Phosphor Stage B Multi Stage C Low Fluorescent Glass Plate Stage and D Fluor Stage are designed to accommodate a variety of sample formats and imaging mode
4. 1 665LP Fig 9 Overlay image of a two dimensional difference gel electrophoresis 2 D DIGE gel with control and treated samples and internal standard The control and treated samples were labeled with Cy3 and Cy5 DIGE Fluors minimal dye labeling protocol The internal standard sample was labeled with Cy2 DIGE Fluor The data sets were evaluated using the DeCyder 2D Software version 7 2 see Fig 10 iCentre i Contest PDGA Cots Cy tn gt ee ee 7 7 a eo N 5 Ks r Os a r a w ri OOO Cold 4 Cyd mm w OSA Calti Cyd mmi 300A CaA Cy3 19 P5GGA Gomi Cya m J i gt t i 9 a lead a Preterm Paie tent amd Av Mathie gt temetent metret th e wre tere 4 r r n f je jes Fig 10 Example of a down regulated protein upon treatment in a DIGE experiment analyzed with DeCyder 2D Software Four control samples left images were compared to four treated samples right images in the Biological Variation Analysis BVA module and a t test was then performed in the BVA workspace The number of detected spots was 2675 and 671 out of them were found to have significant differences at p values lt 0 001 os ay oe f cS ee mera a TF Py ss a i Fig 11 Example of a down regulated protein 2 3 fold decrease upon treatment in a DIGE experiment analyzed with ImageMaster 2D Platinum IMP version 7 software Both 2D a
5. 64 73 BAS IP phosphor screens are recommended for Typhoon FLA scanners The different screens are designed for general use MS high resolution suitable for morphological work such as autoradiography SR detection of the weak energy of the Tritium signal TR and detection of Neutron ND All mounted and unmounted GP phosphor screens except 35x43 mounted GP phosphor screen are compatible with Typhoon FLA 9500 These products can be scanned with a Fluor stage included in the standard configuration Minimum computer requirement OS Windows 7 Professional SP1 32bit Windows XP SP3 32 bit or Windows Vista Business SP2 32 bit RAM more than 1 GB Processo Intel Core 2 Duo processors Hard disk more than 80 GB USB Ports USB 2 0 Optical drive DVD ROM or Super Multi Drive Monitor 1280 x 1024 pixel resolution or higher Please contact your local sales representative for the latest recommended computer configuration 29 0044 13 AB 7 For local office contact information visit www gelifesciences com contact www gelifesciences com quantitative_imaging GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala imagination at work GE imagination at work and GE monogram are trademarks of General Electric Company Amersham Cy CyDye DeCyder Deep Purple ECL ECL Plex Hybond ImageQuant and Typhoon are trademarks of GE Healthcare companies IRDye and LI COR are trademarks of LI COR Biosciences
6. Alexa Fluor SYBR SYPRO and TOTO are trademarks of Molecular Probes Inc Windows Windows XP and Windows Vista are trademarks of Microsoft Corporation Intel and Core are trademarks of Intel Corporation FAM is a trademark of Applera Corporation 2 D DIGE 2 D Fluorescence Difference Gel Electrophoresis 2 D DIGE technology is covered by US patent numbers 6 043 025 6 127 134 and 6 426 190 and equivalent patents and patent applications in other countries and exclusively licensed from Carnegie Mellon University DeCyder This release of DeCyder version 2 software is provided by GE Healthcare to the customer under a non exclusive license and is subject to terms and conditions set out in the 2 D Differential Gel Electrophoresis Technology Access Agreement Customer has no rights to copy or duplicate or amend the Software without the prior written approval of GE Healthcare Deep Purple Total Protein Stain Deep Purple Total Protein Stain is exclusively licensed to GE Healthcare from Fluorotechnics Pty Ltd Deep Purple Total Protein Stain may only be used for applications in life science research Deep Purple is covered under a granted patent in New Zealand entitled Fluorescent Compounds patent number 522291 and equivalent patents and patent applications in other countries CyDye This product or portions thereof is manufactured under an exclusive license from Carnegie Mellon University under US patent number 5 268 486 and equivalent patents
7. GE Healthcare Life Sciences Data file 29 0044 13 AB Imaging systems software and accessories Typhoon FLA 9500 biomolecular imager Typhoon FLA 9500 Fig 1 is a robust and versatile laser scanner that is ideally suited to multiuser environments Biomolecular imaging applications include sensitive and quantitative measurements of Western blots multiplex fluorescence and radioisotopic labels by storage phosphor as well as digitization of colorimetric stains Typhoon FLA 9500 delivers i ot l l Fig 1 Typhoon FLA 9500 is a high performance versatile laser scanner for e Versatility imaging of multifluorescent chemifluorescent sensitive and quantitative measurements in a multiuser environment radioisotope labeled and colorimetric samples e High resolution and quantitation a pixel resolution of The system provides several imaging modes such as up to 10 um and a linear signal response over five orders fluorescence filmless autoradiography and digitization of magnitude provides precise quantitation in gels blots of colorimetrically stained gels For chemiluminescence tissue sections and arrays detection of low abundance proteins the ImageQuant e High sample throughput scanning area of 40 x 46 cm imager series is recommended enables simultaneous imaging of up to 20 gels or blots The system is optimized for differential protein expression measuring 10 x 8 cm in size This facilitates comparisons Studies and quantit
8. The images exhibit low and even background in all three CyDye detection channels which allow for multiplexing applications over a broad linear dynamic range A selection of the dilution series is shown in the image The linear dynamic range of Cy2 labeled carbonic anhydrase was 3 6 orders of magnitude 29 0044 13 AB 3 Carbonic anhydrase ng 409 6 204 8 1024 51 2 256 128 64 3 2 16 08 0 4 0 2 Saan lt lt Sample Carbonic anhydrase 8 Gel 12 acrylamide 70 Tris glycine Imaging Excitation Emission filter 7 Cys 532nm BPG1 570DF20 65 LOD 0 2 ng carbonic anhydrase DR 3 3 orders of magnitude Linearity R 0 998 and slope k 0 99 trendline in log log plot 5 5 log integrated intensity Or 4 5 2 3 4 5 6 log pg carbonic anhydrase Fig 6 A dilution series of carbonic anhydrase in the LMW marker labeled with CyDye DIGE fluor Cy3 minimal dye was separated using 1 D electrophoresis The gel was imaged with Typhoon FLA 9500 The detection limit LOD was 0 2 ng carbonic anhydrase and the linear DR was 3 3 orders of magnitude Arrow indicates the LOD DNA 12 8 6 4 3 2 1 6 0 8 0 4 0 2 0 1 f fmol Sew Sample Cy3 and Cy5 data not shown labeled 81mer oligonucleotide Gel TBE Urea 1 0 mm Imaging Excitation Emission filter Cy3 532 NM BPG1 570DF20 Cy5 635nm LPR 665 LP LOD 0 2 fmol for both Cy3 and Cy5 labeled 81mer Fig 7 A Cy3 labeled 81 mer oligonucleotide was run on a 1 D elect
9. a 4 2 25 3 355 4 45 5 55 log nCi g Fig 12 Scanned image of a C autoradiographic standard using the Typhoon FLA 9500 29 0044 13 AB 5 NIR detection Typhoon FLA 9500 has two PMT s that are optimized for sensitive detection over a broad spectrum from visible light and up to the near infrared NIR region Figure 13 shows how the multialkali PMT 2 gives a linear response and sensitive detection of an IRDye 680 labeled secondary antibody in a Western blot Typhoon FLA 9500 Transferrin 2500 1250 630 310 160 78 39 Sample Transferrin 7 Membrane Hybond LFP 65 Detection Primary antibody o Rabbit anti human 6 transferrin D T Secondary antibody Anti rabbit IRDye 680 5 Imaging Excitation Emission filter Typhoon 685nm BPFR700 R715 3 gt LOD 19 5 pg transferrin 4 DR 2 1 orders of magnitude 1 2 3 4 Linearity R 0 994 and slope k 0 94 log pg transferrin trendline in log log plot Fig 13 A two fold dilution series of transferrin starting at 2 5 ng was subjected to Western blotting and detected with a rabbit anti transferrin primary antibody and anti rabbit IRDye 680 secondary antibody The arrow indicates the LOD 6 29 0044 13 AB 19 5 pg Digitization Excitation light passes through the sample and excites a fluorescent plate The emitted light from the plate passes through the sample again and is collected and converted to an electrical signal The method is suita
10. ative protein detection e g among blots and reduces workload and waiting time multifluorescent Amersham ECL Plex imaging for precise eT ee eee quantitation of two or more proteins in the same Western blot e Flexibility optimized performance for new applications by adapting the system with stages detectors filters and lasers e 2 D DIGE imaging simultaneously image two 2 D DIGE gels for differential expression studies e Visible and infrared fluorescence imaging optional near infrared excitation for imaging IRDye and other infrared dyes Typhoon FLA 9500 is a variable mode laser scanner with modular access to the optical components Fig 2 and 7 excitation sources providing both versatile and flexible imaging for precise quantitation of proteins nucleic acids l i and other biomolecules Table 1 Fig 2 Filters are easily exchanged by the user Broad linear dynamic ra nge Table 1 Typhoon FLA 9500 specifications Typhoon FLA 9500 provides a broad linear dynamic range in Detection modes Fluorescence phosphorimaging all detection modes for example when using Cy 5 labeled digitization and chemiluminescence proteins Fig 3 Excitation wavelengths 473 nm blue LD laser 532 nm green SHG laser 5 635 nm red LD laser 685 nm optional near IR LD laser and 785 nm optional near IR LD laser a Radioisotopes eC EE a og i T and other sources of ionizing radiation E Dynamic range 5 orders
11. ble for documentation of colorimetrically stained gels Data storage Data are stored either in linear 16 bit grayscale TIFF TIF file format or in square root encoded 16 bit TIFF GEL file format The GEL format encoding provides higher dynamic resolution than TIF at lower signal levels to exploit the low signal detection capability of the phosphorimaging technology Image analysis Designed for seamless data transfer and quantitative gel and blot analysis we provide image analysis software for use with Typhoon FLA 9500 Table 3 Table 3 Image analysis software Software Analysis ImageQuant TL 1 D gel electrophoresis dot blots arrays colony counting and user defined gel analysis DeCyder 2D Differential high resolution 2 D DIGE analysis including Extended Data Analysis ImageMaster 2D Platinum 2 D gels including single stain and 2 D DIGE Validation support A comprehensive suite of life cycle validation services is available for laboratory systems used in good practice environments such as GLP GMP or GCP The documentation is developed and approved by validation experts Installation Qualification and Operation Qualification IQ OQ are performed on site by trained service engineers Our engineers can also help with periodic re qualification RQ and evaluate verify and document system changes and software upgrades with Change Control Protocols CCP Ordering information System Quantity Code number
12. d with Typhoon FLA 9500 in separate detection channels The arrow indicates the limit of detection LOD for GAPDH The minimal cross talk and low background enables reliable quantitation of specific signals relative to a housekeeping protein 2 D DIGE The 2 D DIGE system is an integrated solution for accurate quantitation of changes in protein expression Tyohoon FLA 9500 Is a part of 2 D DIGE system that includes DeCyder 2D Differential Analysis Software or ImageMaster 2D Platinum Software Fig 9 10 and 11 The strengths of Typhoon FLA 9500 high sensitivity and broad dynamic range for measuring low and high abundant proteins in one scan Fig 3 make it highly suited for 2 D DIGE applications enabling the user to detect and accurately quantitate subtle changes in protein expression By generating overlaid multichannel images for each gel with minimal crosstalk Tyohoon FLA 9500 exploits the multiplexing potential of CyDye DIGE fluors to remove experimental variation between gels Images are analyzed using DeCyder 2D or ImageMaster 2D Platinum to accurately and confidently measure very small differences in protein abundance Sample 1 Control Cell lysate of human cell line A431 2 Treated Cell lysate of human cell line A431 treated with EGF IPG strips 24 cm 3 11 NL Gel Precast low fluorescent DIGE gel Imaging Excitation Emission filter Cy2 473nm BPB1 530DF20 Cy3 532 nm BPG1 570DF20 Cy5 635 nm DBR
13. e low and high abundant proteins in a single scan Figs 5 6 and 7 Multiple fluorescent wavelengths can be detected with minimal crosstalk for comparative expression experiments See Table 2 for emission filters Table 2 Emission filters Filter type IP LPB 510LP Wavelength range nm BP390 gt 510 Detection examples Phosphorimaging Cy2 SYBR Green FAM FITC Alexa Fluor 488 SYPRO Ruby SYPRO Orange GFP Cy2 DIGE Fluor ECL Plex Cy2 Cy3 Deep Purple HEX Alexa Fluor 532 and 555 SYPRO Red Cy3 DIGE Fluor ECL Plex Cy3 Cy5 Alexa Fluor 633 TOTO 3 DID Cy5 DIGE Fluor ECL Plex Cy5 Alexa Fluor 700 IRDye680 IRDye 700 Alexa Fluor 790 IRDye800 BPB1 530DF20 520 to 540 LPGAS 5LP Zt BPG1 570DF20 560 to 580 LPR 665 LP 2 665 BPFR700 R715 713 to 726 BPFR800 R810 814 to 826 Carbonic anhydrase 167 56 19 6 2 0 7 0 3 0 2 ng Y Sample Carbonic anhydrase in LMW marker 2 8 Gel Amersham ECL Gel 4 20 SDS PAGE 7 tris glycine Imaging Excitation Emission filter g 6 Cy2 473nm BPB1 530DF20 LOD 170 pg z gt DR 3 6 orders of magnitude e Linearity R 0 999 and slope k 0 92 So 4 3 A 5 6 trendline in log log plot log pg carbonic anhydrase Fig 5 The LMW marker proteins were labeled with CyDye DIGE fluor minimal dyes and separated using the Amersham ECL 1 D electrophoresis gel The gel was imaged with Typhoon FLA 9500
14. nd 3D images of the protein of interest Match ID 477 are shown in left side upper and lower respectively Eight control samples upper images were compared to eight treated samples lower images by IMP and the p value and fold time change are presented in the Class Analysis Table in the right upper panel highlighted with green background color A bar chart representative of the down regulation is presented in right lower panel In the DIGE experiment the effect of an activator of protein kinase C PKC Phorbol 12 myristate 13 acetate PMA on the BALB c 3T3 cell was examined The control cells were treated with DMSO This experiment represents a DIGE application on intracellular signal transduction pathway regulated by PKC Detection of radioactivity Samples containing radioactive probes are exposed to a storage phosphor screen Light is emitted from the screen in proportion to the amount of radioactivity in the sample upon laser induced stimulation Fig 12 All GE Healthcare provided storage phosphor screens are compatible with the Typhoon FLA 9500 uCi g 200 70 20 7 2 0 7 0 2 0 07 l Sample C autoradiographic 8 standard CFQ9702 _ 75 Imaging Excitation Emission filter gt FLA 9500 635 nm IP BP 390 z 7 Exposure 60 minutes exposure to ec 65 BAS MS Imaging Plate 9 LOD 0 2 uCi g S N 4 6 Linearity R2 0 999 and slope k 0 97 L oe g 55 trendline in log log plot Fa m 5 O 4 5
15. ns of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Japan Corporation Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 29 0044 13 AB 11 2012
16. of magnitude 2 2 Bit depth 16 bit E Scanning area 40 x 46 cm Pixel sizes 10 25 50 100 200 um and prescan 1000 um a Standard filters IP Phosphorimaging 1 2 3 4 5 6 LPB 510LP LPG 575LP LPR 665LP EEEE E TNT LPR Ch2 665LP BPB1 530DF20 and BPG1 570DF20 ny Seen eee Optional filters BPFR700 R715 BPFR800 R810 DBR1 530DF20 665LP K eee im and DGR1 570DF20 665LP Dimensions W x H x D 900 x 400 x 800 mm n Weight 97 kg Line frequency 50 60 Hz Temperature 15 C to 30 C Sample Carbonic anhydrase in Humidity 20 to 70 no condensation LMW mark Gel Amersham ECL Gel 4 20 Supply voltage 100 240 VAC 10 SDS PAGE tris glycine Imaging Excitation Emission filter Power consumption Approx 0 5 KVA Cy5 635nm LPR 665 LP LOD 25 pg DR 4 3 orders of magnitude o Linearity R 0 997 and slope k 0 96 Technical features trendline in log log plot l o Optimal choice of filter stage laser and PMT Fig 3 Carbonic anhydrase was labeled with CyDye DIGE fluor Cy5 minimal dye and separated using the Amersham ECL 1 D electrophoresis gel The Filters are easily accessed and exchanged without tools to gel was imaged with Typhoon FLA 9500 A selection of a three fold dilution attain optimal imaging conditions This makes the instrument series is shown in the image arrow indicates the limit of detection LOD i i a highly suitable for use in a multiuser environment The
17. rophoresis gel The gel was imaged with Typhoon FLA 9500 The detection limit was 0 2 fmol indicated by the arrow for both the Cy3 and Cy5 labeled oligonucleotides Sensitive multiplex detection of Western blots Typhoon FLA 9500 is a versatile scanner that is ideal for imaging of fluorescent Western Blot membranes This method is very sensitive and the signal is proportional to protein quantity Moreover it is possible to detect more than one protein at the same time by means of secondary antibodies labeled with different fluorophores Amersham ECL Plex fluorescence detection systems provide high sensitivity as well as a broad linear dynamic range and are well adapted to quantitative Western blotting Fig 8 4 29 0044 13 AB HeLa cell lysate 5000 2500 ng total protein 1250 625 313 156 78 39 Sample HeLa cell lysate mixed with transferrin Membrane Hybond LFP Target proteins Transferrin and GAPDH Detection Primary antibodies Rabbit anti human transferrin Mouse anti GAPDH Secondary antibodies ECL Plex Cy5 GAR and Cy3 GAM Imaging Excitation Emission filter Cy3 532 NM BPG1 570DF20 Cy5 635 nm LPR 665 LP LOD GAPDH in 39 ng HeLa cell lysate Fig 8 Multiplex detection of proteins by Amersham ECL Plex Western Blotting Transferrin and endogenous GAPDH were targeted in a dilution series of HeLa cell lysate using ECL Plex anti rabbit Cy5 red and anti mouse Cy3 green secondary antibodies Imaging was performe
18. s Scanning is rapid and detection is sensitive for laser induced fluorescence radioisotopic imaging by storage phosphor and digitization A fast 1000 um prescan function gives a rapid overview of the sample for selecting the correct settings At a pixel size of 200 um a 10 x 15 cm sample is scanned in two minutes The system provides a linear signal response over five orders of magnitude This together with digitization of the image with up to 16 bit resolution provides a suitable basis for the precise quantitation of proteins DNA and other labeled molecules Lasers can be exchanged in the field to accommodate new applications and fluorophores The system can house up to four lasers simultaneously from a choice of five laser excitation wavelengths 473 532 635 685 and 785 nm For the detection of radioactivity and fluorescence emitted light is collected and transformed to an electrical signal by a photomultiplier tube PMT The electrical signal is then converted into digital information by A D conversion for image display and analysis Imaging applications Typhoon FLA 9500 covers all application modes including fluorescence radioisotopes as well as colorimetric stains Flourescence detection Upon excitation light is emitted from a fluorescently labeled sample in proportion to the amount of labeled protein or DNA in the sample The high sensitivity and broad dynamic range of Typhoon FLA 9500 makes it possible to measur
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