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User Manual - GE Healthcare Life Sciences

1. Fig 5 4 Anchor the gel with the gel holders 4 Assemble the gel holders within the slots in the tray Anchor the gel down firmly with the gel holders tightening the screws no more than finger tight Note For large gels the gelholders should be positioned in the geltray before the gel is placed Otherwise the gel could be damaged by the gel holders Fig 5 5 Correct position of gel and reference markers in the gel tray Ettan Spot Picker User Manual 18 1147 61 Edition AA 37 ED Setting up the Spot Picker 5 4 Switch on mains power to the instrument Turn on mains power to the instrument with the mains switch located on the back of the instrument 5 5 Start Ettan Spot Picker Control software 1 To start the Ettan Spot Picker Instrument Control Software select Programs Ettan Spot Picker Ettan Spot Picker from the Windows Start menu e Ettan oe 1 Spot Picker qye e Version 1 00 M De Lo gt gt AR A or 3 s 4 db i amersham pharmacia biotech amp 2 A dialogue regarding Homing of instrument will be shown Press OK Homing Before continuing the system must be homed Press OK to start homing Cancel WARNING MOVING PARTS The picker head and camera assembly can make sudden rapid movements Keep all body parts clear when the Spot Picker is in operation 38 Ettan S
2. 300 c 6 The pick list data will appear on screen and a representation of the spots to be picked will be drawn in the main part of the window X and Y coordinate columns are empty because the reference markers have not been detected yet Elton Spot Picker Be System ods bos 1 7121 6342 1263 799 3 1206 ens 4 1002 5 1507 126 6 1556 11065 8 1611 7799 3 1035 11 1013414 1515 1119281 6585 12 19579 5738 13 19507 5696 14 10644 7611 15 1947 10311 16 15867 1208 17 1625 1183 18 1458 12411 19 17005 1966 18102 12760 N 19411 1257 2 296 1283 3 11660 1000 M 11420 9790 aar 606 JE H Fig 6 2 Loaded picklist 7 In the Load Pick List window press Next Ettan Spot Picker User Manual 18 1147 61 Edition AA 51 6 Picking a gel 52 9 If microplates have not been previously loaded label the appropriate number of microplates as shown on screen and load the first four or fewer onto the Spot Picker microplate rack See Section 5 2 Ifa gel has not been loaded place the gel according to Section 5 3 Load Gel And MicroPlates Prepare 1 Labelled MicroPlate lt Back Next gt Press Next 6 3 Detection of the reference markers 1 2 In the Find First Marker IR1 window press Move to First Marker Move the camera with the blue arrows to a position over the first reference marker IR1 on the gel O lt Set Co
3. of the Bind Silane solution onto the plate and distribute equally over the plate with a lint free tissue Cover the plate to prevent dust contamination and leave to air dry on the bench for 1 1 5 hours 6 Polish the plate with a lint free tissue moistened with a small amount of double distilled water or ethanol The gels will stay attached to the glass during electrophoresis staining procedures scanning and storage 3 3 Positioning of the reference markers Spot picking with the Ettan Spot Picker requires the use of reference markers The reference markers are used to enable the Spot Picker to transform image pixel X Y co ordinates for each spot into a millimetre position to pick from This enables picking from gels where the protein spots are fluorescently labelled as well as from gels where the proteins have been stained with a visible stain The reference markers are designed so that they will be visible on the scanned image of a gel fluorescent or visible protein stain as well as being visible to the marker detection camera on the Ettan Spot Picker Two reference markers must be included in each gel that is to be picked from The reference markers are self adhesive and should be attached to the gel backing glass plate or GelBond film prior to gel casting or at the latest before the scanning of the gel The reference markers can be used in both densitometric and fluorescence scanning Ettan Spot Picker User Manual 18 1147 6
4. oida em 5 To export data press the Export Data button Open File Export Data Exit 6 Save the data as a text file which may then be used in other applications such as Microsoft Word and Microsoft Excel 62 Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel without a pick is N 7 Picking a gel without a pick list 7 1 Introduction The Ettan Spot Picker allows spot picking in a mode similar to manually picking the spots This feature is useful when there are a limited number of spots to pick The gel does not need to be scanned and no pick list must be prepared The picking will not produce a result file so the user must keep track of the spots Note Use the delivered white sheet as a background to obtain a better contrast of the gel image in the camera view 7 2 Manual picking directly from gel without a pick list The Ettan Spot Picker gel and microplates must have been prepared according to Chapter 5 Setting up the Spot Picker The function uses the picking parameters as set in the System setup Therefore these need to be set correctly before using Click n Pick 1 Press Initialise Instrument and Go to Home 2 Select Tools Click n Pick lt Set Contrast gt lt Set Brightness gt z 7 UKI x a 7 7 m 7 Exit 3 Select the Picker Head Size This will alter the size of the circle in the camera view window Ettan Spot Picker User Manual 18 1147 61 Edition
5. AA 63 Picking a gel without a pick list The first gel plug will be dispensed in microplate well A1 However the position can be edited in the Next Well window Set Contrast Brightness and camera aperture to achieve a good image quality Move the square in the upper right window with the mouse pointer to a preferred position of the gel Move the circle in the camera view by using the mouse pointer When a requested gel spot is detected within the picker circle press Pick Tip It may be convenient to place e g a pen close to the spot which will help to find it in the camera view Do not activate the movements of the picker during this procedure Note When the picker head has dispensed the gel plug into the micro plate the camera will move back to the picked position WARNING MOVING PARTS The picker head and camera assembly can make sudden rapid movements Keep all body parts clear when the Spot Picker is in operation 8 9 If several gel plugs from a spot are wanted to be pooled in the same microplate well it is possible by checking the Dispense in Single Well box in the Next Well window This has to be done before the first picking to that well When the microplate is full Replace the plate with an empty plate and press New Plate 10 When the picking run is completed Press Exit 64 Ettan Spot Picker User Manual 18 1147 61 Edition AA 8 Troubleshooting Troubleshooting CI To avoid error
6. D manua proteomics Ettan Spot Picker User Manual O am 18 1147 61 amersham pharmacia biotech Important user information All users must read this entire manual to fully understand the safe use of Ettan Spot Picker WARNING A The Warning sign highlights an instruction that must be strictly followed in order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Caution The Caution sign is used to call attention to instructions or conditions that must be followed to avoid damage to the product or other equipment Be sure not to proceed until the instructions are clearly understood and all stated conditions are met Declaration of conformity Safety standards This product meets the requirements of the Low Voltage Directive 72 23 EEC through the harmonized standard EN 61 010 1 1993 A2 1995 EMC standards This product meets the requirements of the EMC Directive 89 336 EEC through the harmonized standards EN 50081 1 and EN 50082 1 WARNING This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adequate measures The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a stand alone unit or connected to other CE marked Amersham Pharmacia Biotech instruments or
7. Fig 3 3 Position of the reference markers below the gel backing Note When attaching the reference markers on a gel backing after the gel casting or electrophoresis run the surface of the backing must be completely dry to ensure good adhesion of the markers 3 4 Gel casting Now assemble the gel holder for casting as described in the manufacturers instructions Pour the gels as soon as they are correctly assembled Ettan Spot Picker User Manual 18 1147 61 Edition AA Preparing the gel 3 5 Gel running conditions The gel being attached to a backing will not affect the standard gel running conditions for a given sample However it is important that the Immobiline DryStrip is correctly orientated The Immobiline Dry strip should be placed as normal e g acidic end to the left with the gel backing lower most 3 6 Gel staining To find examples of fixing and staining protocols see Chapter 9 Staining protocols 3 7 Gel scanning with the ImageScanner 3 7 1 Gel position In order to secure the targeting accuracy of the Ettan Spot Picker it is of particular importance to place the markers in the correct position during scanning e The markers must be placed in the middle of the scanning area Fig 3 4 e The top of the gel which was in contact with the Immobiline Dry strip must be placed on the right side when looking at the scanner as showed in Fig 3 4 e The gels must be scanned upside down with the gel in conta
8. a gel 58 6 9 Repeating a picking run In case of unsuccessfully picked gel plugs it is possible to repeat a run to complete the picking The picking parameters may need to be changed e g increase the jazz 1 2 3 Note the positions of microplates and wells with missing gel plugs In the Load Pick List window press Load Pick List Browse for the result of the performed picking run extension mfl Select the result file Do not use the correction function again Click on Open Sy Results Pick list Resultmf Scanner Correction T Use Correction Scanner ID Sample Cancel Resolution DPI 200 z a The result table will be shown on the screen SpotNo ImageX ImageY PickerX PickerY Plate No Y 52 7 86 0 Well y 3 4 123 4 34 6 5 169 4 89 4 6 77 4 146 5 7 24 0 173 9 8 111 1 208 0 3 128 7 135 0 0 51 7 52 8 NI ENT ENIES ENKI EN ENIES Y Y Y Y Y Y Y VV VV a DDD I I I I Note The detection of reference markers has to be done again The Picker X and Y values are missing in the table 6 Perform detection of reference marker see Section 6 3 Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel CI es 7 In the updated result table select the rows for each unpicked gel plug and the ticks in the table will be replaced by filled circles 8 Press Pick The user will be informed by the software to place the specific pla
9. connected to other products recommended or described in this manual and used in the same state as it was delivered from Amersham Pharmacia Biotech except for alterations described in this manual All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them A copy of these terms and conditions is available on request Ettan ImageMaster ImageScanner PlusOne Hoefer GelBond Immobiline and Typhoon are trademarks of Amersham Pharmacia Biotech Limited Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia amp Upjohn Inc Gilson is a trademark of Gilson Inc Coomassie is a trademark of Imperial Chemical Industries PLC Decon is a trademark of Decon Laboratories Limited SYPRO is a trademark of Molecular Probes Inc Microsoft Microsoft Excel Microsoft Word and Windows NT are trademarks of Microsoft Corp Amersham Pharmacia Biotech AB 2000 All rights reserved Office Addresses Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech Inc 800 Centennial Avenue P O Box 1327 Piscataway N J 08855 1327 USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Pharmacia Biotech KK Sank
10. event of an emergency press the Stop button on the Ettan Spot Picker front panel The instrument immediately ceases movement and all motors are de energized 2 4 2 Power failure routine In the event of a power failure the experiment is interrupted in an undefined state However the data for gel plugs picked up to the power interruption is saved When power returns the following will happen 1 The picker head and the syringe pump dilutor are homed 2 The PC starts and displays the log in dialogue 2 4 3 Restart Procedure In the event of system shutdown due to power failure emergency stop or process interruption malfunctions must be rectified before the Ettan Spot Picker is restarted To restart the Ettan Spot Picker 1 Check that the Power indicator on the Ettan Spot Picker instrument lights 2 Select Programs Ettan Spot Picker Ettan Spot Picker from the Windows Start menu Reload the resultfile and continue picking see instructions in Section 6 10 The last picked gel plug may be lost This depends on the exact moment when the interruption occurred Ettan Spot Picker User Manual 18 1147 61 Edition AA Preparing the gel 3 Preparing the gel 3 1 Introduction Spot picking with the Ettan Spot Picker requires that gels are precast on backing e g Ettan DALT II Pre cast Gel 12 5 or immobilized on backing during casting Two different types of backing may be used either GelBond PAG film or a glass plate trea
11. gel twice in ddH O for five minutes each rinse 5 Incubate the gel in Developing solution for up to 10 minutes If the staining is intense enough before the end of 10 minutes move directly to the stop solution 6 Pour the developing solution off and add the Stop solution incubate for 10 minutes 7 Rinse the gel three times in ddH O for five minutes each rinse 8 Store the gel in ddH O 9 4 2 Using the Hoefer automatic gel stainer The following method was developed for 1 mm thick 12 acrylamide gels Other thickness and percentage acrylamide gels may require further optimization The wash steps which follow the sensitizing and silver stages are crucial for a low background to the gel Make all solutions freshly when needed Make sure to use only double distilled 18 2 MQ water All solutions should appear clear and colourless before they are poured onto the gel Reagent Quantity Sodium thiosulphate 500 mg ddH 0 To a final volume of 250 ml Table 9 5 Sodium thiosulphate solution 0 2 Ettan Spot Picker User Manual 18 1147 61 Edition AA 71 ED Staining protocols 72 Reagent Quantity Glacial acetic ethanoic acid 12 5 ml ddH 0 To a final volume of 250 ml Table 9 6 Stop solution Fix the gel for 2 hours minimum in 30 ethanol 7 5 acetic acid Wash the gel four times in 50 methanol for eight minutes each rinse Sensitize for 30 minutes in 0 2 sodium thiosulphate Wash the gel five times in ddH O
12. may be weakened if other operations are performed Ettan Spot Picker User Manual 18 1147 61 Edition AA 15 ED Safety instructions 2 2 Safety labels The following safety labels are attached to the Ettan Spot Picker instrument to warn the user of potentially hazardous conditions At the picker head Fig 2 1 Safety label located on the Y arm near the picker head WARNING MOVING PARTS The picker head and camera assembly can make sudden rapid movements Keep all body parts clear when the Spot Picker is in operation WARNING Never put your hands underneath the picker head during instrument operation The picker head has the capacity to puncture skin In the event of an emergency press the Stop button on the Spot Picker the instrument immediately ceases movement and all motors are de energized WARNING To prevent fire or shock hazard do not spill liquids into the camera body 16 Ettan Spot Picker User Manual 18 1147 61 Edition AA Safety instructions ECE 2 3 Other warnings WARNING HEAVY OBJECT The Ettan Spot Picker weighs about 40 kg Two persons are required to lift the instrument WARNING NO SERVICEABLE PARTS INSIDE Do not open covers Service and maintenance should be performed by qualified personnel Ettan Spot Picker User Manual 18 1147 61 Edition AA 17 FR Safety instructions 18 2 4 Emergency procedures 2 4 1 Emergency shutdown In the
13. plug from It is therefore very important that spots are accurately detected so that the correct area of gel is picked The following points should be considered during spot selection for picking e The dimension of the picker head limits picking of spots very close to each other It is advisable to get an idea of the size of the picker head in the gel image by using a pen tool of appropriate size Fig 4 1 Ettan Spot Picker User Manual 18 1147 61 Edition AA 27 ED Selecting spots on the gel and creating a pick list 28 e It may be preferable to manually specify the picking position for spots with irregular or long drawn out shapes and for very large spots Fig 4 1 The use of Draw Spots pen tool to simulate the size of the picker head By using the Draw Spots tool an approximate size of the picker head on the scanned image can be seen For a gel image which was scanned at 300 dpi a pen size of 17 will represent a 1 4 mm diameter picker head Similarly for a gel image scanned at 300 dpi pen size 24 corresponds to a 2 0 mm picker head 4 1 2 Detection and editing of the markers The two reference markers included in the gel must also be detected as spots Determining the centre of the marker is very important for accurate picking Often the reference markers are not accurately detected with the parameters used for detecting the protein spots in your image If the reference markers have not been correctly detected the
14. plugs have been located e When a run has been stopped Ettan Spot Picker User Manual 18 1147 61 Edition AA 49 6 Picking a gel AA File System Tools About File None Spot No ImageX ImageY Pickerx Picker Plate No Well 4 No Pick List Loaded Move X Y No AAA listloaded Load Pick List i penal White Markers Black Markers lt SetContrast gt lt Set Threshold gt Intialize Instument Marker Ofset 007 N ToHome Marker Y Dlf set 0 07 ssa Fig 6 1 Main window before loading a pick list Before proceeding it is advisable to press the Initialize Instrument and Go to Home buttons 6 2 Load a pick list 1 In the Load Pick List window select the type of used reference markers Black White Press the Load Pick List button r Load Pick List Select Marker Type White Markers Black Markers Load Pick List Mert A 50 Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel CI Browse for the pick list 3 Select the pick list 4 If the gel has been scanned with an ImageScanner check the Use Correction box Select the used scanner in the Scanner Id list box and select the used resolution when the gel was scanned in the Resolution DPI list box 5 Click on Open C C List Sc gt 2 ake E ake2 E ake3 GJ ake4 Y COMPAQ a dmi Pick List Jake txt Scanner Correction M Use Correction Resolution DPI
15. the reference markers on the gel backing outside The gel fescue mmm kasan samaa iy ts 22 Position of the reference markers below the gel backing 22 Correct positioning of the gel on the ImageScanner 24 Orientation of gel image before SaviNg oooccccccooocccccconooncnnncnnnna 26 The use of Draw Spots pen tool to simulate the size Of The piekerhead us sus Mairin Sama cudehenceMbnchcwetstedee eae 28 Correctly detected reference Marker ouusunnnnn onneen 29 Incorrectly detected reference markers oooooooooccnnnnnccncnnnccnonononos 29 Correct appearance of the Measurements Window 33 Correct table format and headings in EXxcel uuuussnnnnnnn 34 Correct pick list format and headings cccccccccccccnnncnnnnonanananns 34 Place the microplate racks ccconnnnnococononoooononanonononononnononcononoss 35 Place the Micro plates ooo Aaa 35 Place Sel trays ita ia Jats lies Veni een eset Nas 36 Anchor the gel with the gel holders cccccccccccccnnnonininnnannos 37 Correct position of gel and reference markers in the gel tray 37 The System Set up WINndow oooccccccnooccnconononcnnnnnncononcnnncnonnanonons 40 Setting the picker head height a Picker head is too high b Picker head is correct height Cp Picker Head 18100 Westin 42 Picking parameters er v0 eet dr aio 43 Example of effect of jazz amplitude on the picki
16. to resume picker movement 6 6 Stop picking Press the Stop button of the instrument or the Stop button in the main application window if the Spot Picker needs to be immediately stopped Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel GI There is a facility to restart a picking run after pressing Stop This procedure is described in section 6 10 6 7 Changing microplates If a picking run consists of more than 384 gel plugs the software will instruct the user to change plates 1 3 Remove the four full plates and cover them with plastic lid or adhesive foil cover Place the reguired number of labelled microplates onto the Spot Picker Press New Plate This event will happen every time four full microplates are left on the spot picker 6 8 After picking When the picking run is completed 1 2 Remove the gel from the gel tray and store discard as appropriate If the gel is to be discarded scrape the gel off the glass plate with a plastic spacer or similar object Then put the plates into a 5 Decon solution overnight to remove any gel fragments which adhere to the plate Rinse the buffer lines with ddH 0 using the prime syringe tool Rinse the liquid out of the gel tray with ddH 0 and leave the gel tray to dry Exit the Ettan Spot Picker Instrument Control Software and turn the computer and the Spot Picker off Ettan Spot Picker User Manual 18 1147 61 Edition AA 57 6 Picking
17. 0 Resume picking after Ettan Spot Picker has been stopped 59 6 11 Open result files ooooocnnnccccccnnnnooonanonocononanononononnnnnccononanonoss 61 Ettan Spot Picker User Manual 18 1147 61 Fdition AA Contents 7 Picking a gel without a pick list Lal s o A en Sed EE amanda T n s 63 7 2 Manual picking directly from gel without a pick list 63 8 Troubleshooting 9 Staining protocols 2 16 lt GOUTIXING asses Bis O 69 9 20 SY PRO TUD cat Aida 69 9 3 SY RRO Orange iia da 69 9 4 Silver staining compatible with MS analysis uuuooossi 70 9 4 1 Using the Modified PlUSONE method susossssninnlli 70 9 4 2 Using the Hoefer automatic gel stainer uuuuuusshslin 71 915 Coomassie stalling ati 72 Ettan Spot Picker User Manual 18 1147 61 Edition AA vil Contents viii Ettan Spot Picker User Manual 18 1147 61 Edition AA Fig 1 1 Fig 3 1 Fig 3 2 Fig 3 3 Fig 3 4 Fig 3 5 Fig 4 1 Fig 4 2 Fig 4 3 Fig 4 4 Fig 4 5 Fig 4 6 Fig 5 1 Fig 5 2 Fig 5 3 Fig 5 4 Fig 5 5 Fig 5 6 Fig 5 7 Fig 5 8 Fig 5 9 Fig 5 10 Fig 5 11 Fig 6 1 Fig 6 2 Fig 6 3 Fig 6 4 List of figures The Ettan Spot Picker SYStOM ccccccccsssssseceseceseceeeeeceeeeeeeeeees 12 Position of the reference markers on the gel backing when gel has been Cast ccccccccncncnonannoonononanononnnnononononnananincnos 21 Position of
18. 1 Edition AA Preparing the gel 3 3 1 Positioning before gel casting It is important that the markers are appropriately placed on the gel backing The markers should be placed according to the following protocol Take care not to place the markers where they will interfere with the pattern of protein spots in the gel 1 Place the spacers on the edge of the treated glass plate GelBond to ensure that the spacers do not cover the markers 2 Place the marker approximately half way along this edge away from the spacer but not so far as to interfere with the protein spot pattern 3 Repeat steps 1 and 2 for the other edge of the gel backing 4 When finished the markers should be in positions similar to those shown in Fig 3 1 Ld Fig 3 1 Position of the reference markers on the gel backing when gel has been cast Ettan Spot Picker User Manual 18 1147 61 Edition AA 21 Preparing the gel 22 3 3 2 Positioning before gel scanning If there is a sufficient backing outside the gel area for example Hoefer SE 600 gels then it is possible to attach the reference markers after casting just before scanning es Fig 3 2 Position of the reference markers on the gel backing outside the gel If there is not a sufficient backing area outside the gel it is possible to attach the reference markers below the backing before gel scanning This is the recommended procedure for Ettan DALT II Pre cast Gel 12 5 E
19. 1147 61 Edition AA Safety instructions ECE 2 Safety instructions IMPORTANT To avoid any risk of injury the instrument should only be operated by properly trained personnel and always in accordance with the instructions provided The purpose of this chapter is to describe safety precautions and present all safety labels that are attached to the instrument Built in safety functions are described and emergency and disposal procedures are included 2 1 Safety precautions 1 Read this entire manual before using the Ettan Spot Picker instrument 2 This instrument is designed for indoor use only 3 The instrument must always be used with the protective earth lead of the power cord correctly grounded to earth at the mains outlet 4 To permit sufficient cooling ensure that the vents at the top and bottom of the instrument are not covered 5 Do not operate the instrument in extreme humidity above 95 Avoid condensation by letting the unit equilibrate to ambient temperature 6 Do not operate the instrument in direct sunlight 7 Keep the instrument dry and clean Wipe regularly with a soft damp tissue Let the instrument dry completely before use 8 Any equipment connected to the instrument should meet the requirements of the EN 61 010 1 or other international safety standards WARNING Do not use the Ettan Spot Picker in any other way than described in this User Manual The protection provided by the instrument
20. ED Selecting spots on the gel and creating a pick list A 4 13 Detection and selection of spots for picking After performing spot detection and spot editing ImageMaster generates a measurements table containing spot related data When the measurements table is saved as a pick list all the spots in the table will be picked Therefore if only a selection of the spots are requested to be picked they have to be defined This can either be done manually or by using the spot filtering tools Manual selection of spots to be picked Once the spot editing is satisfactory go to the spot filtering mode 1 Deselect all spots by pressing the Select Deselect all button All the spots should appear blue If they do not press the button again 2 Select the spots for picking by clicking on them with the left mouse button 3 The reference markers must also be selected 4 Incase of errors click on the spot with the right mouse button to deselect the spot 5 Once this process is finished see Section 4 1 5 Creating the pick list Automated selection of spots to be picked Once the spot editing and spot matching are satisfactory they must be complete to apply the automatic filtering go to the spot filtering mode Decide to apply either comparison or range parameters If parameters are set in both comparison and range they will both be applied when the Apply Filter button is pressed no matter which window is displayed e The Range parameter
21. Master 2D Elite gel image analysis software A pair of reference markers must be included within the gel and also detected during image analysis These reference markers are used to enable the Spot Picker to transform image pixel X Y co ordinates for each spot into a millimetre position to pick from Following excision from the gel the plugs are dispensed into 96 well microplates along with a volume of dispensing fluid The Ettan Spot Picker is capable of picking up to 9600 plugs in any single picking run 1 1 The Ettan Spot Picker system The Ettan Spot Picker system comprises the following e Ettan Spot Picker instrument e Ettan Spot Picker Instrument Control Software running under Windows NT operating system on a PC e PC with frame grabber card for video display Fig 1 1 shows the Ettan Spot Picker system A detailed description of the Ettan Spot Picker instrument is provided in the Ettan Spot Picker Instrument Handbook Ettan Spot Picker User Manual 18 1147 61 Edition AA 11 ED Introduction 12 Fig 1 1 The Ettan Spot Picker system 1 2 Method overview With the Ettan Spot Picker gel plug picking can be defined in a number of steps 1 Preparing and pouring the gel onto a backing including reference markers Alternatively use precast gels and include the reference markers before scanning Running the 2D separation Fixing and staining the gel after electrophoresis Scanning the gel including applie
22. R referemce markers mms ssite sis A ii 20 CESISMALINGS ia ada 31 CGELECTION savossa whence ioe TR SURRAR these ities PaSa e ity 52 incorrect detection ita ida dci 55 positioning before gel casting uuuuuussnnnm nnam 21 positioning before gel scanning oooooccccccnicoccciconionanancnonnnnns 22 reference markers area sooouunumioii nnas 47 result files EXPOR A MA KAS am Vee cas nS ASTMAA tee rak 62 lOCATION A annus iit ansa Nm 56 O NN 61 PINSE SELOKES etarra td ta oei 46 ASENCIO dad A RS 46 74 Ettan Spot Picker User Manual 18 1147 61 Edition AA S safety EN T SNES TS ERS TEATER sm Te AT TN ee SAR ST eaten ace 16 other Warnings lt a a 16 PIECAULIONS rta data tio 15 Scanner Correction cunas ii blade 51 selecting spots auto Mat A 30 lmageMaster vensa ieor da aA E EE ATETEA 27 manual e ta dle eo cd 30 SIIVErStaI NINE dada dead 70 Hoefer automatic gel stainer coccincococccccconooccnccononnncnononon 71 Plus Ohne method 00 da dadas TRENN 70 SYPRO orange sta ning ii tds 69 SYPROWuby Staining cai 69 SUSIEMESCIUD rra ai da ltd siorse S t 40 T threshold iia tii a L A ia 52 TYPHOON O COPE OO en 13 26 Ettan Spot Picker User Manual 18 1147 61 Edition AA 75 76 Ettan Spot Picker User Manual 18 1147 61 Edition AA
23. Setting up the Spot Picker The Dispense Vol should be large enough to ensure that each gel plug is successfully ejected from the picker head If this value is set too low then gel plugs will not be dispensed into the microplate and will be lost when the picker head is next rinsed The dispense volume should not be below 100 ul Dispensing success rate 10 15 20 30 40 50 60 70 100 dispensing volume ul Fig 5 11 Example of effect of dispensing volume on the dispensing success rate A 1 4 x 1 2 picking head was used to pick from 1mm thick gels 12 T on glass and GelBond backing The Dispense Flow needs to be high enough to rapidly eject the plug from the picker head It is recommended that the dispense flow value is set to max 30 ml min 5 8 5 Lift The Lift option allows you to raise the picker head from the gel backing prior to aspirating the gel plug into the head Under normal conditions no lift is reguired In some cases introducing a lift may actually reduce the picking efficiency 5 8 6 Rinse volume and Rinse strokes The Rinse Volume and Rinse Strokes parameters determine the efficiency of picker head washing after each gel plug is picked Under normal conditions a single syringe stroke with a volume 100 950 ul is sufficient Using more rinse strokes will increase the length of the picking runs 46 Ettan Spot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker 5 5 9 Referenc
24. ab Set the Co ordinate field to Pixels Click OK Activate the Measurements Window Using the Select Fields button make sure that only the Spot Scaled Coords Pixels and Comment fields are selected Deselect any other column by un ticking the appropriate box If the order of the columns left to right does not appear the same as in Fig 4 4 use the Move Up and Move Down buttons in the field selection box until they are correctly ordered Ettan Spot Picker User Manual 18 1147 61 Edition AA Selecting spots on the gel and creating a pick list EN A 6 The position of the reference markers spots 1 amp 14 in Fig 4 4 in the pick list is unimportant 383 Measurements Window FIEF E3 13 47 1105 869 Fig 4 4 Correct appearance of the Measurements Window 4 1 6 Saving the pick list file When the measurement window table is correctly formatted the pick list can be saved 1 With the Measurements Window active select Edit Copy To File 2 Enter a suitable file name for the pick list 3 The pick list must be saved as a text txt file No further editing of the pick list should be done at this point If any errors are found in the pick list it is preferable to go back to the appropriate step and create a new pick list 4 2 Creating pick list files from alternative gel image evaluation softwares Ettan Spot Picker Instrument Control Software can not handle original pick list data from alternative 2D gel imag
25. acetic acid 3 Stain the gel for three hours overnight with gentle shaking protected from the light 4 Wash the gel in 7 5 acetic acid for 60 90 minutes Ettan Spot Picker User Manual 18 1147 61 Edition AA 69 ED Staining protocols 9 4 Silver staining compatible with MS analysis 9 4 1 Using the Modified PlusOne method Allow at least 250 ml of each solution per gel All steps are carried out at room temperature with gentle shaking Make all solutions freshly when needed Make sure to use only double distilled 18 2 MQ water All solutions should appear clear and colourless before they are poured onto the gel Reagent Quantity Ethanol 75 ml 5 Sodium thiosulphate 10 ml solution Sodium acetate 17g ddH 0 To a final volume of 250 ml Table 9 1 Sensitizing solution Reagent Quantity Silver nitrate 625 mg ddH 0 To a final volume of 250 ml Table 9 2 Silver nitrate solution 0 25 Reagent Quantity Sodium carbonate 6 25 g Formaldehyde 100 ul ddH 0 To a final volume of 250 ml Table 9 3 Developing solution 70 Ettan Spot Picker User Manual 18 1147 61 Fdition AA Staining protocols CI Reagent Quantity EDTA 3 65 g ddH 0 To a final volume of 250 ml Table 9 4 Stop solution 1 Sensitize the gel in Sensitizing solution for 30 min Rinse the gel three times in ddH O for five minutes each rinse Incubate in a 0 25 silver nitrate solution for 20 minutes RW N Rinse the
26. at the picking precision will not be affected Ettan Spot Picker User Manual 18 1147 61 Edition AA 53 6 Picking a gel 54 11 12 13 14 15 16 17 18 19 When the offset values are in the range 0 1 to 0 1 press the Move to First Marker button Note It is only when the Move to First Marker button is pressed that the picker records the position of the marker Press Next In the Find Second Marker IR2 window press Move to Second Marker Move the camera with the blue arrows to a position over the second reference marker IR2 on the gel When the perimeter of the Marker detection window is green the picker has detected a stable image of the reference marker If the reference marker is not visible in any of the windows or the perimeter is red see the step 3 7 Press the Adjust button and the reference marker will be centered in the Marker detection window The marker is assumed to be centred when the values in both offset boxes are in the range 0 1 to 0 1 The procedure may need to be repeated until the offset values fall within the acceptable range When the offset values are in the range 0 1 to 0 1 press the Move to Second marker button In the Find Second Marker IR2 window press Next The software will now calculate the coordinates for the spots to be picked and the pick list table on the screen will be updated Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel CI A No
27. ct with the glass surface of the scanner with a thin layer of water or gel storage liquid between The liquid layer will also make it easier to lift the gel after scanning Ettan Spot Picker User Manual 18 1147 61 Edition AA 23 Preparing the gel Fig 3 4 Correct positioning of the gel on the ImageScanner 1 Pourafew mlof gel storage liguid or water onto the glass surface of the scanner 2 Hold the gel on backing at an angle over the scanning surface 3 To avoid air bubbles slowly lower the gel until it is flat and resting on the scanning surface 4 To avoid seeing water drops on the gel image wipe any drops off from the upper surface of the gel backing with a lint free tissue 24 Ettan Spot Picker User Manual 18 1147 61 Edition AA Preparing the gel em 3 7 2 Scanning 1 Inthe ImageMaster Labscan software select the function Scan a new image 2 Select Refresh preview 3 Select the scan area with the mouse arrow Start in the lower left position of the gel image on the screen Drag an area to the upper right position of the preview area even if this area is not showing the gel Note Itis crucial to maximize the selected scan area in the upper right position before releasing the mouse button Otherwise the calibration of the ImageScanner will be affected and resulting in imprecise spot picking 4 Select the resolution 300 dpi and Scan mode transmissive 5 Start scan 6 After scanning press the Rotat
28. d reference markers Analysis of the gel image selecting the protein spots to pick and creating the pick list file Loading the gel onto the Ettan Spot Picker and excising the selected gel plugs into microplates Ettan Spot Picker User Manual 18 1147 61 Edition AA Introduction GI 1 3 The User Manual The Ettan Spot Picker User Manual provides technical information and operating instructions for the Ettan Spot Picker instrument and the Ettan Spot Picker Instrument Control Software Gel casting and gel running are described briefly In addition instructions for scanners and gel image evaluation softwares are included For detailed information on the instrument the user is referred to the Ettan Spot Picker Instrument Handbook 1 4 Accessories In addition to the items delivered with the system Ettan Spot Picker reguires the following accessories Gel scanner ImageScanner or Typhoon e Gel image evaluation software ImageMaster 2D Elite e 96 well microplates See the Ettan Spot Picker Instrument Handbook for ordering information 1 5 Associated documentation The Ettan Spot Picker Instrument Handbook provides safety instructions technical information and basic operating instructions for the Ettan Spot Picker instrument In addition maintenance schedules and instructions for user maintenance are included Ettan Spot Picker User Manual 18 1147 61 Edition AA 13 1 Introduction 14 Ettan Spot Picker User Manual 18
29. e Marker area The reference markers area is set in the Marker Area section of the System Setup window The two values minimum and maximum stipulate the size in pixels of the reference markers These two values are used during detection of the reference markers to evaluate the objects seen by the camera The default values for these parameters are Min Area 30 000 and Max Area 46 000 Min Area 10000 95000 30000 Marker Area in pixels Max Area 15000 100000 46000 5 10 Save and Exit System Setup Click on Save amp Exit when the system configuration is completed The same settings will be used until altered by the user Ettan Spot Picker User Manual 18 1147 61 Edition AA 47 ED Setting up the Spot Picker 48 Ettan Spot Picker User Manual 18 1147 61 Fdition AA Picking a gel CI 6 Picking a gel 6 1 Introduction For a successful picking run it is assumed that all the necessary preceding steps have been carried out in accordance with the instructions in this User Manual and Ettan Spot Picker Instrument Handbook A gel is now placed into the picker and a pick list has been created This section describes the actions to perform before during and after picking Loading of a pick list Detection of the reference markers Start picking Stop pause picking Changing microplates Procedure after picking Handling of result files The section also describes the resuming of picking runs e When unpicked gel
30. e evaluation softwares However if the required data can be exported to Microsoft Excel it will be possible to use the data for spot picking with Ettan Spot Picker Ettan Spot Picker User Manual 18 1147 61 Edition AA 33 ED Selecting spots on the gel and creating a pick list To create a pick list from an alternative 2D gel image evaluation software 1 Export the data to Microsoft Excel The following data are required e Spot identity e The co ordinates of the centre of the spots e The co ordinates must be in pixels e The x and y co ordinates must have separate columns 2 Edit the data in Excel to create a table in a correct format e The table must contain only four columns e The headings of the columns must be as follow id x y comment e The comment column must contain the IR1 and IR2 annotations for the reference markers If the 2D gel image evaluation software does not allow exporting designations the IR1 and IR2 annotations must be assigned manually to the appropriate spots id x y comment 1 10 1001R1 2 12 4 1000 3 30 5001R2 Fig 4 5 Correct table format and headings in Excel 3 Save the file File Save as as a Tab delimited text txt file File name Jpickiist txt v Save as type Text Tab delimited txt X mi id X y comment 1 10 100 IR1 2 12 4 1000 3 30 500 IR2 Fig 4 6 Correct pick list format and headings 34 Ettan Spot Picker User Manual 18 1147 61 Fdition AA Setting up the Spo
31. e inner side of the picker head is dirty Clean the picker head both inner and outer sides Gel from spot has been picked but is not found in microplate well Gel plug is situated in a nearby well The microplate Z height has been too high so the dispense flow has splashed it into a nearby well Decrease the microplate Z height Gel plug is in rinse station due to adsorption to the outer side of the picker head during dispensing The dispensing height has been set too low Increase the microplate Z height Clean the outer side of the picker head to reduce liquid adsorption Gel plug is in rinse station due to adsorption to the outer side of the picker head during dispensing The dispensing volume has been set too low Increase the dispensing volume Clean the outer side of the picker head to reduce liquid adsorption Gel plug is lost during transfer from gel to microplate Increase the aspiration volume Very imprecise picking results The identities of the two reference markers have been mixed up Confirm that the first detected marker is the correct one on the gel image 66 Ettan Spot Picker User Manual 18 1147 61 Edition AA Troubleshooting CI Problem Probable cause Remedy Picking position errors Camera is not correctly calibrated Check that the calibration has been performed according to the Instrument Handbook Incorrect detectio
32. en Building 3 25 1 Hyakunincho Shinjuku ku Tokyo Japan Contents 1 Introduction 1 1 The Ettan Spot Picker system ooocccccncoconnnccoconononononononnnnnncnonnnass 11 T2 Method OVe VIEW inutil 12 LS MEUS Mantal ii Ia A a NR ERT 13 1 45 MACCESSORIES am im ira 13 1 5 Associated documentation ossmmsssesrererrrrrrerrrrrrrsrsssrrrrrrrr renar snara annan 13 2 Safety Zool Safety Pprecatitions Er 15 22 Safety labels ea it ciendo Es 16 29 OHO Warnings area nettet ned Ba Ht oe ies 16 2 4 Emergency procedures c cooooccnccncconcnnncnnnoonnnnnnnononnnnnnnonononnnnnonininos 17 2 4 1 Emergency SHUTDOWN curia tala aa ahistaa IZ 2 4 2 Power failure routine is sa iris 17 2 4 3 Restart Procede odas lees de 17 3 Preparing the gel SEL INTOA A maan E AINEENA STANS SY PL AE ETT 19 3 2 Bind Silane treating glass plates cccccoconcccncooocccccncononannnnnnnn 19 3 3 Positioning of the reference markers mnsssssssrrrrssssrrrrressererrsrnrrr rn nn 20 3 31 Positioning before gel CaSting oocccocoococcnocooaannconnonanannnonnn 21 3 3 2 Positioning before gel SCANNING ccccnccccocoooaooooanonanononononononoss 22 MA O AL ge en 22 3 5 Gel running conditions mosmsssessrrrersrsrsrsseseserenenerererrer enes rr rss narr nonan 23 326 gt Gel STAINING enviarla ad KA ANN KEN LAS 23 3 7 Gel scanning with the ImageScanner munsmeeeesserrrsrrsrsrssrsrsrnniinn 23 3 7 1 G POSITION sen a ech A oh ae ai 23 3 12 SCANNING vassa rissa la Ae LS A
33. for 20 minutes each rinse Incubate in 0 25 silver nitrate solution Table 9 2 for 30 minutes Wash the gel twice in ddH O for four minutes each rinse Incubate in developing solution Table 9 3 for 2 5 minutes Incubate in Stop solution Table 9 6 for 60 minutes Store the gel in ddH O 9 5 Coomassie staining 1 2 Fix the gel for 60 minutes in 30 ethanol and 7 5 acetic acid Stain the gel for 60 minutes minimum in 0 1 Coomassie R 250 in 40 ethanol and 10 acetic acid Use a Stock solution of 1 w vol Coomassie R 250 in 96 ethanol Destain the gel in 20 ethanol and 5 acetic acid Store the gel in ddH O Ettan Spot Picker User Manual 18 1147 61 Edition AA AAA Index A aspirate volea a o do a a id aa 44 ASMA tried 45 B Bind Silane treatment sms is sissi a AN fear ing r da aa 19 c Gamera Offset mito rta ita outs neg ETARE EEG 53 Camera VIEW WINGOW arica a a i 52 Contrast ar a am s As a aH ETAT a ah ek 52 COOMASSIE STAINING ssssiasaastan ta italianas 72 D dispense TOW eco a iii 46 di PENSO VOIUME a a eee 46 E Ettan DALT Il Pre cast Gel 12 5 ooooconococonococcnoncnanannnncnnnnncronnnos 19 Ettan Spot Picker compatibility o ooooocconccccccccononnnonocononanos 11 Ettan Spot Picker Instrument Control Software oi 11 Stat tht ER SENAT LAKKO A cate cen aches 38 Ettan Spot Picker Instrument Handbook unmnmmssssrsrssrsrsrssssrrrsrra 13 Ettan Spot P
34. fore saving the image the image on the screen has to be in the orientation with the acidic pH to the left basic pH to the right and low Mw at the lower end Rotate the image to the specified position and save the image pH y Mw Fig 3 5 Orientation of gel image before saving Ettan Spot Picker User Manual 18 1147 61 Edition AA Selecting spots on the gel and creating a pick list EN 4 Selecting spots on the gel and creating a pick list 4 1 Using ImageMaster 2D Elite The basis of spot picking with the Ettan Spot Picker is a pick list created by an image analysis software This section describes how to create this pick list using ImageMaster 2D Elite V3 00 and higher Essentially the pick list needs to contain the location in pixels of the centre of each spot you wish to pick It must also contain the pixel co ordinates of the centres of the two reference markers This section assumes that the user is familiar with at least basic spot detection and editing using ImageMaster For more detailed information on the use of ImageMaster consult the User Manual 4 1 1 Simulating the picker head size When a spot boundary has been detected by ImageMaster or drawn by the user it is assigned a position in the image in terms of X and Y pixel co ordinates This corresponds to the centroid of the spot as defined by the spot boundary These are the co ordinates used to define the position that the picker head will remove a gel
35. gels and picker heads have been compiled Gel Picker Recommended parameters head Thickness Backing Type Jazz Asp vol Asp flow Disp vol Disp flow Lift 1 mm glass 14x1 2 0 9 1 1 15 50 20 100 250 30 0 2 0x 1 2 1 0 1 2 30 50 20 150 250 30 0 GelBond 1 4x 1 2 0 7 1 0 15 50 20 100 250 30 0 2 0x 1 2 1 1 1 3 30 50 20 150 250 30 0 1 5 mm glass 1 4x 1 7 1 2 1 4 25 50 20 100 250 30 0 2 0x 1 7 1 2 1 4 25 50 20 150 250 30 0 Table 5 1 Recommended picking parameters 5 8 2 Jazz The Jazz parameter determines the amount of sideways movement that the picker head will perform at its lowest Z position to shear the gel plug from the gel backing A Jazz of 1 mm corresponds to a movement of 1 mm both in the plus and minus direction along the X axis of the spot picker Ettan Spot Picker User Manual 18 1147 61 Edition AA 43 ED Setting up the Spot Picker The amplitude of the Jazz depends primarily on the picker head size and is also dependent on e Type of backing e Quality of the Bind Silane treatment e Percentage of polyacrylamide used e Gel thickness Spot picking success rate 100 3 0 0 4 0 5 06 0 7 0 8 09 1 1 1 jazz mm Fig 5 9 Example of effect of jazz amplitude on the picking success rate A 1 4 x 1 2 picker head was used to pick from 1mm thick gels 12 T on glass backing 5 8 3 Aspirate volume and Aspiration flow The function of aspirating is to suck u
36. icker system ssooouinnnnm nanain 11 G gel image evaluation software occccnnocccnccncooonnnnnnnononnnnnononnnnnos 13 gel running conditions esaesa darik n aae enee A 23 A A eaa i aa a ten rN 13 ES A k a N 23 TYPROON T wr steokccun tanion tol aE 26 GOI As ssd dont saline le Sd Pa HTA U as META Soule dann SUSAN VAAN STT STUKA ees 36 GeIBONG PAG AIIM toda 19 I MageMaSie ta a ds 11 13 27 lmageScanner cccccococcccnncononnnnnnnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonnnanos 135 223 J Jaz na emt hanat o o Math dd ete at ter ce 43 L IT E acces teens tn ce tect est cake bast RK E E aa 46 ees Ettan Spot Picker User Manual 18 1147 61 Edition AA 73 DANS POWER Hai teman Pa eco edad tas ea 38 MALDI TOF E dador e de 11 MANUAL PICKING caida dada 63 marker detection WINdOW ccccccccncnnnnononononononannononnnnononnnnnonoss 52 MICrO Platessa ssa saa ii a 35 Microplate ack ams ds thal dd ahaa vai 35 P pick list CREATING A DIS KN Ue te be A aa ds alan att Mestre 32 from alternative gel image evaluation softwares 33 A E EOI 50 teseneratihg nyn meann ao a and eA aan 58 SVIN OA Ags e A r a a RING 33 picker head Selec ehen a a ar ea Abst 35 simulating SIZE amma n bid 27 SIZE CI CIS AA ed a eS 63 Z POSILION VSBEl irc nia tito 41 picking SE OA 56 TES UG dd a dido 59 A AE TE 56 STO O tal uted 56 PICKING parameters o de da ovina ad did 43 precast gels samma id a 12 PMA a A a RESA E ac aaa 39
37. icro plate Pick Date Dig State _ Spot Date User 3 1 1 187 5 158 3 1131 227 8 31 08 00 4 2 1 187 5 316 6 127 8 227 8 31 08 00 5 3 1 187 5 475 1425 227 7 31 08 00 6 4 1 187 5 633 3 157 2 227 6 31 08 00 7 5 1 187 5 791 6 171 9 227 6 31 08 00 8 6 1 187 5 950 186 6 227 5 31 08 00 g T 1 187 5 1108 201 3 227 5 31 08 00 10 8 1 187 5 1266 216 227 4 31 08 00 11 3 1 187 5 1425 230 7 227 4 31 08 00 12 10 1 187 5 1583 245 4 227 3 31 08 00 13 11 1 187 5 1741 2601 227 3 31 08 00 14 12 1 187 5 1900 2748 227 2 31 08 00 15 13 1 375 158 3 113 2104 31 08 00 16 14 1 375 316 6 127 7 210 3 31 08 00 17 15 1 375 475 1424 210 3 31 08 00 18 16 1 375 633 3 157 1 210 2 31 08 00 19 17 1 375 791 6 171 8 210 2 31 08 00 20 18 1 375 950 186 5 210 1 31 08 00 21 19 1 375 1108 201 2 210 1 31 08 00 22 20 1 375 1266 215 9 210 31 08 00 2 21 1 375 1425 230 6 210 31 08 00 24 22 1 375 1583 245 3 209 9 31 08 00 25 23 1 375 1741 260 209 9 31 08 00 26 24 1 375 1300 274 7 209 8 31 08 00 2 25 1 5625 158 3 113 193 31 08 00 28 26 1 562 5 316 6 127 7 1929 31 08 00 29 27 1 562 5 475 1424 192 9 31 08 00 30 28 1 562 5 633 3 157 1 1928 31 08 00 31 23 1 562 5 731 6 171 8 1928 31 08 00 32 30 1 562 5 50 186 5 192 7 31 08 00 33 31 1 562 5 1108 201 2 1927 31 08 00 34 32 1 562 5 1266 215 9 1926 31 08 00 35 3 1 562 5 1425 230 6 1926 31 08 00 36 34 1 562 5 1583 245 3 1925 31 08 00 37 35 1 562 5 1741 260 1925 31 08 00
38. ig 5 7a the tip of the picker head is not touching the gel backing This will result in the gel plugs being left in the gel or picked partially If a gap greater than 2 mm can be seen Fig 5 7c the picker head is pressing down too hard on the gel backing This will result in a shorter life span for the picker head and has the potential to scratch glass plates The correct position for the picker head is with a 1 2 mm gap showing at the top of the piston Fig 5 7b Press the Set Gel Z Coordinate when the Z position of the picker head is correct Ettan Spot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker 5 EE saki 5 8 Picking parameters There are various parameters that must be set correctly for successful picking and dispensing of gel plugs It is difficult to give exact values for some of them because the optimal values may differ depending on types of backing percentage gel and staining methods The parameters can be varied in experiments to optimize the picking from different gel types Picking Parameters gt Jazz 0 2 mm Aspirate Vol 0 100 ut 20 Aspiration Flow 1 30 ml min f20 Dispense Vol 0 300 ul 100 Dispense Flow 1 30 ml min 30 Lift 0 10 mm o 7 Rinse Volume 0 950 ul 500 Rinse Strokes 1 20 1 Fig 5 8 Picking parameters 5 8 1 Recommended parameters Out of performed experiments some guidelines and recommendations for different type of
39. ion tool button and rotate the image counter clockwise once The resulting image will show the gel with the acidic pH to the left basic pH to the right and low Mw at the lower end 7 Save the image as a tif file with File Save as Note To secure the picking accuracy of the Spot Picker no other manipulations with the gel image is allowed e g rotating or resizing Ettan Spot Picker User Manual 18 1147 61 Edition AA 25 Preparing the gel 26 3 8 Gel scanning with the Typhoon It is important to place the gel directly onto the scanner plate Do not reassemble The gel must be scanned upside down with the gel in contact with the glass surface of the scanner with a thin layer of water or gel storage liquid between The liquid layer will also make it easier to lift the gel after scanning 1 Pour a few ml of gel storage liquid or water onto the glass surface of the scanner 2 Hold the gel on backing at an angle over the scanning surface 3 To avoid air bubbles slowly lower the gel until it is flat and resting on the scanning surface 3 8 1 SYPRO ruby stained gels It is recommended to use the green laser 532 nm and the emission filter of 610 nm with 30 nm bandpass Use the platen focus setting 3 8 2 SYPRO orange stained gels The green laser 532 nm is recommended The emission filter of 580 nm with 30 nm bandpass should be selected Use the platen focus setting 3 8 3 Image on screen after scanning Be
40. ker X and Y values are missing in the table 6 Perform detection of reference marker see Section 6 3 7 Press Pick The user will be informed by the software to place the microplates The partially dispensed micro plate and the next undispensed microplates must be placed in their original positions G INFO 007 Place microplate s Place microplates on the instrument Put plate 3 at position 3 E He CE 60 Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel CI Note Depending on exactly when picking was interrupted the last gel plug being picked may be lost 6 11 Open result files The format for the file names is Directory name XX apb where the directory name is the same as the name entered in the Output Directory Name field and the XX is the number of the plate produced in that picking run 1 To view result files associated with each microplate of gel plugs select Tools Output File Viewer 2 To open a plate file press the Open File button in the lower end of the dialogue Export Data Exit 3 Browse and select the file to be viewed and click Open Open Output File 12 x Look in ja result003 y E el E File name resutto03 001 apb Files of type Output Filef apb Cancel T Open as read only Ettan Spot Picker User Manual 18 1147 61 Edition AA 61 Picking a gel E 4 The viewer allows the user to look at the processing status of the gel plugs in that m
41. m the same area Storage of the gel in double distilled water reduces the risk of cracking Ettan Spot Picker User Manual 18 1147 61 Edition AA 67 ED Troubleshooting 68 Ettan Spot Picker User Manual 18 1147 61 Edition AA Staining protocols CI 9 Staining protocols 9 1 Gel fixing When the gel run is complete the gel must be placed into a suitable fixing solution as soon as possible This will minimize protein spot diffusion As the gel is attached to a backing a fix solution that dehydrates the gel too quickly will cause the gel to crack and peel away from the backing It is recommended that a solution of 30 ethanol and 7 5 acetic ethanoic acid is used to fix gels The gels must be left in this fix solution for at least two hours Leaving gels in this fix for longer periods of time appears to have no detrimental effects on the gel 9 2 SYPRO ruby 1 Fix the gel in 10 methanol and 7 acetic acid for at least 2 hours 2 Place the gel directly into a polypropylene polycarbonate or polyvinyl chloride tray 3 Cover the gel with the SYPRO ruby stain 4 Incubate the gel for five hours overnight with gentle shaking protected from the light 5 Pour off the SYPRO ruby 6 Wash the gel in 10 methanol 6 acetic acid for 1 2 hours 9 3 SYPRO orange 1 Place the gel directly into a polypropylene polycarbonate or polyvinyl chloride tray 2 Prepare 250 ml of a 1 5000 dilution of SYPRO orange in 7 5
42. n follow these steps to define the reference markers Alternative 1 e Use the Grow Peaks tool and click inside the reference marker Alternative 2 1 Select a pen size slightly smaller than the size of the reference marker 2 Draw a spot on the reference marker placing it centrally on the marker 3 Use the Grow Edge tool to fit the spot boundary to the boundary of the marker Ettan Spot Picker User Manual 18 1147 61 Edition AA Selecting spots on the gel and creating a pick list EN Note For accurate spot picking it is essential that the reference markers are correctly detected When the edges of the markers are not properly detected the position on the pick list given for the centre of the marker will be incorrect This will lead to an incorrect transformation of the spot co ordinates from the pick list into mm positions for the picker to pick from Make sure that the position reference markers were detected as distinct circular spots and that the outline of the spot well corresponds to the outline of the marker This is illustrated in Fig 4 2 Fig 4 2 Correctly detected reference marker Drops of water below and above the gel during the scanning can affect the detection as seen in Fig 4 3 2 An imprecise use of the detection tools can also result in an incorrect detection see Fig 4 3 3 Fig 4 3 Incorrectly detected reference markers Ettan Spot Picker User Manual 18 1147 61 Edition AA 29
43. n of the reference markers Be sure to have a distinct circular picture of the marker both in the camera and marker detection view Uncorrected ImageScanner image Scan according to User Manual and use the correction option when the pick list is imported There is no calibration file for the used scanner Contact APBiotech Service for calibration of the scanner The gel image has too low resolution Scan the gel with higher resolution for example 300 dpi Picking errors for oval and irregular shaped spots The centre of the detected spot in the 2D software differs from the preferred picking position Detect these types of spots manually For example with the Grow Peaks tool or the Pen tool Scratches on the gel glass plates after picking The Z height of the picker head versus gel is too low Adjust the Z height according to User Manual The gel loosens from the glass plate Inefficient Bind Silane treatment Prepare according to recommendations in User Manual Clean the plates properly and leave them in 5 Decon over night The gel loosens from the glass plate in a specific area during picking Too many gel plugs have been picked from a smaller area The gel cracks and become damaged close to the picked gel plug Gel stored in ethanol and acetic acid has a tendency to crack This will not affect the picking result if not too many spots are picked fro
44. n of the picker head according to microplates and rinse station For instructions see the Ettan Spot Picker Instrument Handbook The height of the picker head versus the gel backing material e The parameters used for picking the gel plugs e The reference markers area ranges which are used by the detection software At any stage the user can decide to abandon the System Setup procedure by pressing the Cancel button If the Cancel button is pressed any changes made to the System Setup will be lost To retain new System Setup parameters press the Save amp Exit button It is advisable to press the Initialize Instrument and Go to Home buttons prior to setting any of the system locations Ettan Spot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker 5 The blue arrows visible on the right hand side of the screen are used to manoeuvre the picker head to various positions Once the picker head neat is placed appropriately e g in the rinse station the location can be stored by pressing the appropriate Set button The blue arrows are used to move the picker head in the X Y Z directions By pressing the buttons on the arrows the head will move stepwise in the corresponding directions As long as a button is pressed ouent F the head will keep moving Current Y mm 124 Hg For the Move X Y arrow the outermost buttons will move the head in 10 mm steps the middle buttons in 1 mm steps and the innermost but
45. nd side reference marker 6 Enter IR2 into the Value field and press Set 7 Press Done BH Image Window oppethusgel3b 2 52 1 ioj x etlerla nea Gel oppethusgel3b Spot 5 Field Commet z Value a I Set Value in all matched spots Ettan Spot Picker User Manual 18 1147 61 Edition AA 31 ED Selecting spots on the gel and creating a pick list ee 8 To view these comments on screen go to Tools Options select the Display tab and tick the Data annotations box and set the field to Comment Check that IR1 and IR2 are in the Comment column in the Measurements window Note When using ImageScanner it is strongly recommended that reference markers are assigned in the manner described above This way reference marker 1 will always be on the left hand side of the gel in the geltray if the Immobiline Dry strip is at the top and marker 2 will always be on the right This will greatly aid identification of the different markers 4 1 5 Creating the pick list The pick list is created from the measurements window However before the pick list can be saved the measurements table must be put in the correct format as described below If the pick list is not saved in this format the picker will not recognise the file and will therefore not be able to pick the spots To create the pick list in the correct format 1 32 Select Tools Options When the options window appears select the Display t
46. ng success rate A 1 4 x 1 2 picker head was used to pick from 1mm thick gels 12 T ON glass DACKINS 4 veces fH Aye tes Florence adie ees va ve NRA Dt dk PSN aaa dete 44 Example of effect of aspirate volume on the picking success rate A 1 4x 1 2 picker head was used to pick from 1mm thick gels 12 T OREA PACKING ta aan ida 45 Example of effect of dispensing volume on the dispensing success rate A 1 4 x 1 2 picking head was used to pick from 1mm thick gels 12 T on glass and GelBond backing ooonooconinccccinocccoccnccconnos 46 Main window before loading a pick liSt ocococnccccccnnnnns 50 Loaded pickli Eur a ri re rate 51 MOVE CAMA a HAT aad 52 Incorrect reference marker detection ssessseresssosererennrn rar 55 Ettan Spot Picker User Manual 18 1147 61 Edition AA ix List of figures x Ettan Spot Picker User Manual 18 1147 61 Edition AA Introduction GI 1 Introduction The Ettan Spot Picker is designed to automatically excise polyacrylamide gel plugs containing proteins from 2D gels and then transfer the plugs to microplates These gel plugs are suitable for further processing prior to MALDI TOF mass spectrometry analysis The Spot Picker is compatible with 2D gels that have been post stained with Coomassie silver or SYPRO dyes and for pre labelled proteins Gels must be immobilized on plastic backing or glass Spots of interest can be evaluated and selected by using the Image
47. ntrast gt lt Set Threshold gt _ Initiaize Instrument Marker X Off set 10 20 Adjust Go To Home Marker Y Off set 0 00 Au Fig 6 3 Move camera 3 If the reference marker is underneath the camera but not visible in the Camera view window use the Contrast and Threshold sliding bars to find an image of good guality If the image appears blurry adjust the camera aperture and focus until the marker image is sharp in the Camera view If the marker is visible in the Camera view window but there is no image in the Marker detection window first adjust the positions of the Contrast and Threshold sliding bars until an image is visible in the Marker detection window If adjusting the sliding bars does not lead to an image becoming visible in the Marker detection window set the Contrast and Threshold sliding bars to their central positions Then Ettan Spot Picker User Manual 18 1147 61 Edition AA Picking a gel CI adjust the camera aperture until the marker is visible in the Marker detection window 5 When the majority or all of the reference marker is displayed in the Marker detection window the perimeter of the window will change from red to green This indicates that there is a stable detection of the reference marker Go to step 9 6 Ifthe majority or all of the reference marker is visible in the Camera view window and the perimeter of the Marker detection window remains red then make sure that the Select Marker T
48. ooooooccccccocccccnncnnnnnnos 38 5 6 Prime the Systemi jssak vs ia tmv ta kv i bsr 39 SS A in Saa etna setae IDKA NY Reis KARTOR SKEN 40 5 7 1 Picker head versus gel Z position cooooococccccoconanonoconnnnnnoos 41 5 8 PICKING parameters oanrin ser asia sr ke kkr Kn detoa a ENA KE pin na Nr bn nono 43 5 8 1 Recommended parameters ccccccccccnnnnononononananonanonnnnononoss 43 5 8 2 JAZZ Pamela mau lettas saa KT alae suc A ATi Kassa 43 5 8 3 Aspirate volume and Aspiration flOW srerrrrrerrrrrrrrrrrrrrrrrn rna 44 5 8 4 Dispense volume and DISPENSE TIOW s messrsrrrssssssrsrrrrrrrnrrnr ran 45 5 8 5 LIE stat mata ainut SN vaa assrai ial E iene 46 5 8 6 Rinse volume and Rinse strokes msnrrrssrrrrrrssrrrrrrsrsrrnrrrnrrrnr nn 46 5 9 Reference Marker area cosuosoosnn io ea U naa enaa annen nan 47 5 10 Save and Exit System Setup ccoooooccnccnoconcnnnonononnonnnnnnonnnanonononnnnns 47 Picking a gel Gl INTO UCA in cae inde ein ere Ti ee dd 49 6 2 Odd as PICK MISE L t o aon st r NL act TK DAA Ak 50 6 3 Detection of the reference markers m ssmmsssrsssrsssserranerrsrrnrennnnannn nan 52 6 4 Rick INES lek cases feeds a ida 56 6 57 Pause PICKING ati rd bd na rr 56 6 6 STOP PICKING Svanin sec evade LA eka his tere leia 56 6 7 Changing microplates isvs ssiesst saamassa mad cove e Sea Aer KI RK 57 6 82 Alter PICKING sil ead iia 57 6 9 Repeating a picking TUN iiisovsnsesnsn ansat iceehetasceeieesesvedeedeeeetes 58 6 1
49. p the gel plug and keep it in place inside the picker head tip prior to dispensing into the microplate The Aspirate Vol should be large enough to create an adequate negative pressure in the inside of the picker head Values between 15 and 50 ul have been found to work very effectively If this value is set too low then the gel plugs will be left behind in the gel Values that are too high may lead to some damage of the gel plugs 44 Ettan Spot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker GG Spot picking success rate AO foeennttttttttaaammmmm m tt aae SN aspirate volume ul Fig 5 10 Example of effect of aspirate volume A 1 4 x 1 2 picker head was used to pick from backing on the picking success rate 1mm thick gels 12 T on glass The Aspiration Flow determines the rate of aspiration A value of 20 ml min has been shown to work effectively If this value is set too low then the gel plugs will be left behind in the gel lead to some damage of the gel plugs Values that are too high may 5 8 4 Dispense volume and Dispense flow Each gel plug is ejected from the picker head along with a volume of fluid The fluid serves two purposes Firstly it physically forces the gel plug out of the picker head Secondly if a suitable buffer is used this can form part of the gel plug washing destaining procedure Ettan Spot Picker User Manual 18 1147 61 Edition AA 45 ED
50. pot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker GG 5 6 Prime the system Place the buffer tubing in a suitable buffer for spotpicking When the picklist is loaded the software will inform the user of the volume of buffer reguired to pick all spots It is recommended that either double distilled water or the appropriate wash destain solution for the gel plugs is used as the system buffer Priming the syringe removes air bubbles in the spot picker tubing and also rinses the tubing with fresh solution Priming should be made when tubing bottles or buffer have been changed Note It is important to ensure that the position of the rinse station is correctly set in the System Setup see Ettan Spot Picker Instrument Handbook before priming the system 1 Select Tools Prime Syringe 2 In the Prime Syringe window enter the number of priming strokes 5 10 priming strokes are recommended Press Prime Priming Strokes B Press Prime tostat Stop Prime Exit 3 The Stop button can be used to interrupt the priming 4 When the priming is completed press Exit Ettan Spot Picker User Manual 18 1147 61 Edition AA 39 ED Setting up the Spot Picker 5 7 System set up 40 Select System System Setup System Setup gm lt ET 50 TA fees n Fig 5 6 The System Set up window The System Setup screen allows the user to set The positio
51. s and to obtain maximum picking success rate it is important to follow all the instructions in the User Manual and Instrument Handbook The user must ensure that e The picker head is not damaged and is clean on both the inner and outer sides e The O ring is present and not damaged e Tubing is clean and not damaged e The Z height values are correctly set e The System setup parameters are correctly set Problem Probable cause Remedy Gel plugs not removed from gel The set jazz amplitude is too low Inefficient aspiration Increase the jazz value stepwise with 0 1 mm increments The aspiration volume should be minimum 15 ul Increase the aspiration volume Check the O ring of the picker head for damage Check all tubing for leaks and air bubbles Bind Silane treatment was too effective Reduce the concentration of Bind Silane The picker head does not reach the gel Check the picker head s Z height versus gel The picker head is worn out Replace the picker head A position close to the current gel plug s has previously been picked Ettan Spot Picker User Manual 18 1147 61 Edition AA 65 ED Troubleshooting Problem Probable cause Remedy Gel plugs are picked but deformed or not complete Only the upper part is picked due to the picker head not reaching the gel backing Adjust the picker head versus gel Z position Th
52. s specify explicit ranges of values that a spot must have in its measurement fields in order to be selected e The Comparisons parameters select spots based on comparisons with spots that are matched to it in other gels For a detailed explanation of how to use these functions please refer to the ImageMaster reference manual and or help function 30 Ettan Spot Picker User Manual 18 1147 61 Edition AA Selecting spots on the gel and creating a pick list EN After the automatic filter has been applied the reference markers have to be selected manually if they have not been automatically selected At this stage it possible to manually edit the spot selections as described in the preceding section Once this process is finished see Section 4 1 5 Creating the pick list 4 1 4 Designating the reference markers The reference markers must be distinguished from protein spots in the pick list This is achieved in ImageMaster by putting an entry into the comment field of the reference markers 1 Inthe main image window select Edit Edit Spot Fields 2 Select the arrow tool and click on the left hand side reference marker 3 In the Edit Spot Fields dialogue box select the Field as comment 4 Enter IR1 uppercase letters with no space between into the Value field and press Set This designates that the co ordinates from this spot represent the first reference marker 5 With the selected arrow tool click on the right ha
53. smut 25 3 8 Gel scanning with the Typhoon sssuuuuunnmnom nannaa 26 3 8 1 SYPRO ruby stained gels ococcccooconocococonanaconononanonaconcnanonaos 26 3 8 2 SYPRO orange stained gelS ccocoooococinocooooncconocannnnononccnnnnass 26 3 8 3 Image on screen after scanning ccccooconccccnoooaanonononenannninnnon 26 Ettan Spot Picker User Manual 18 1147 61 Edition AA Vv Contents vi Selecting spots on the gel and creating a pick list 4 1 Using ImageMaster 2D Elite uossnsssssrsrrsssserersssssrrrsssrrerrsrnrrrr renen rna 27 4 1 1 Simulating the picker head size occoccoocccconocooaonncononanananos 27 4 1 2 Detection and editing of the markers ccuuuuussssssnnnnsnnn 28 4 1 3 Detection and selection of spots for picking 30 4 1 4 Designating the reference MarkeFS ocooooococccncoocaancnnconnnnnnonos 31 4 1 5 Creating the pick liSt cccccccccccccccessccccceesssscsecseestssseecestssseees 32 4 1 6 Saving the pick list fileira a 33 4 2 Creating pick list files from alternative gel image evaluation sofiWakes vasama redden aos va in 33 Setting up the Spot Picker 5 1 Selectspicker Ahead iia Al San 35 52 Placesthe cmicro plates d eo PT Tm Mind bees N Wea esas 35 15 37 Place THE gel st rs eel ss anlecdesvienieeumsiinstnae a an tele 36 5 4 Switch on mains power to the instrument coccnnnnncccconcoooonnnnnnnnns 38 5 5 Start Ettan Spot Picker Control software cc
54. t Picker 5 5 Setting up the Spot Picker 5 1 Select picker head If the picker head needs to be changed see instructions in the Ettan Spot Picker Instrument Handbook 5 2 Place the microplates 1 Place the microplate racks in the Spot Picker Fig 5 1 Place the microplate racks 2 Place 1 4 microplates in the plate tray with A1 position in the front Fig 5 2 Place the microplates Ettan Spot Picker User Manual 18 1147 61 Edition AA 35 ED Setting up the Spot Picker 5 3 Place the gel 1 Place the gel tray in the Ettan Spot Picker The guide feet on the tray should fit onto pins on the locator plate it is only possible to fit the gel tray in one direction Fig 5 3 Place gel tray 2 Place the gel in the gel tray and cover with a suitable fluid e g fixing preservative liquid or double distilled water to a surface above the gel of approximately 2 mm If many spots will be picked a larger volume of fluid may be required to compensate for evaporation 36 Ettan Spot Picker User Manual 18 1147 61 Edition AA Setting up the Spot Picker G Note To avoid air bubbles place fluid in the tray before the gel The gel is then placed in the tray angled to the buffer surface 3 Position the gel so that the reference markers lie within the parallel lines in the centre of the gel tray see Fig 5 5 The reference markers must not cross or touch the lines because this will affect the reference marker detection
55. te Damaged reference markers and impurities in the gel image can result in incorrect detections Also reflections shadows and incorrect set contrasts thresholds and camera aperture can result in similar errors This will result in imprecise picking Move XZY lt Set Contrast gt lt Set Threshold gt Initialize Instrument Marker X Off set n Joi Adjust Go To Home Marker Y Off set 0 29 ad Fig 6 4 Incorrect reference marker detection Ettan Spot Picker User Manual 18 1147 61 Edition AA 55 6 Picking a gel 6 4 Pick the gel 56 1 2 In the Pick Spots window press Pick In the Result File Location window select the location for the picking results file s 3 Enter output directory name and user name The results will be saved as a folder in the selected location Each plate filled even if only partially during the picking run is given a unique results file Result File Location E ake4 E COMPAQ GC dmi Y Gilson 5 HySnapDX x AClient cfg a AClient dat Ake txt CO REM INI yl Output Directory Name ake5 User Name ake Create Directory amp Start Picking po 4 Click on Create Directory amp Start Picking 6 5 Pause picking Press the Pause button if the picking procedure has to be paused to fill the buffer reservoir for example When the Pause button is pressed the picker will pause at the end of a specific operation Press the Continue button
56. te in position 1 Note Itis important to remove the volumes of liquid in the empty wells otherwise the volume will be too high after re picking G INFO 007 Place microplate s 9 Press OK and the picking run will start 6 10 Resume picking after Ettan Spot Picker has been stopped 1 When the Ettan Spot Picker has been stopped by user or due to a technical error an information message appears Press OK G INFO 025 Picking finished The picking has stopped due to an error The instrument will now be re initialized To recover load the result file and restart 2 In the Load Pick List window press Load Pick List Ettan Spot Picker User Manual 18 1147 61 Edition AA 59 6 Picking a gel Browse for the result of the interrupted picking run extension mfl 4 Select the result file Do not use the correction function again Click on Open Mikael 3 Results Pick list Resultmil Scanner Correction TF Use Correction Scanner ID Sample v S Resolution DPI 300 5 The result table will be shown on the screen Ticked positions mean picked gel plugs Filled circles represent unpicked gel plugs Y 3 46 9 146 2 Y 4 46 9 292 3 Y 5 46 9 438 5 e 6 46 9 584 6 e 7 46 9 730 8 e 8 46 9 876 9 e 3 469 1023 1 e 10 469 11692 e 11 469 13154 Note The detection of reference markers has to be done again The Pic
57. ted with a Bind Silane solution Note To scan a gel with fluorescently labelled proteins it is important that GelBond is not used as the gel backing The reason for this is that GelBond is a plastic material and all plastics have intense fluorescenses at the wavelengths used for scanning 3 2 Bind Silane treating glass plates The following protocol for treatment of glass plates was optimized for Bind Silane from Amersham Pharmacia Biotech Note It is important that glass plates are properly clean Before re use soak the plates overnight ina 5 Decon 90 solution Do not leave plates standing in a Decon solution for a longer time as this will eventually cause etching due to the alkali nature of Decon 1 Thoroughly wash the plate to be treated Take care to remove any gel fragments attached to the plate from previous gels The careful cleaning of the glass plates before casting is important to ensure a uniform coating with the Bind Silane and to avoid keratin contamination 2 Thoroughly rinse the plates with ddH O to remove Decon 3 Dry the plate using a lint free tissue or leave them to air dry 4 Prepare the Bind Silane working solution see Table 3 1 Ettan Spot Picker User Manual 18 1147 61 Edition AA 19 Preparing the gel 20 Reagent Quantity Ethanol 8 ml Glacial Acetic acid 200 ul Bind Silane 10 ul Double distilled HO 1 8 ml Table 3 1 Bind Silane working solution 5 Pipette 2 4 ml depending on plate size
58. tons in 0 1 mm steps The Move Z arrow buttons will move the head in 5 1 and 0 1 mm steps respectively Current Z mm 175 0 _Tntsse Instrument CAUTION Be careful when moving the picker head because if the head hits an obstacle the motor will shut off and the instrument will be re initialized Similarly rapidly moving the picker head into an object has the potential to damage the picker head 5 7 1 Picker head versus gel Z position This value indicates the Z height at which the picker head meets the gel backing This must be correctly set for maximum picking efficiency Note This value must be checked before every picking run To set the correct Z height of the picker head 1 Use the Move X Y blue arrows to move the picker head to an area where there is gel backing but no gel or no important area of the gel 2 Use the Move Z arrows to carefully lower the picker head Using the innermost button will move the picker head in 0 1mm steps Ettan Spot Picker User Manual 18 1147 61 Edition AA 41 ED Setting up the Spot Picker 42 3 When the tip of the picker head touches the backing you will see a gap between the holder and the picker head piston y i E TFT y Fig 5 7 Setting the picker head height a Picker head is too high b Picker head is correct height c Picker head is too low 4 If a gap does not form F
59. ype box in the Load Pick List window has the correct colour of marker selected Use the Back button to go to the Load Pick List window Use the Next button to return to the Find First Marker IR1 window When the perimeter is green go to step 9 7 Tf there is a portion or none of the reference marker visible in the Marker detection window and the perimeter of the Marker detection window is red this means that the camera is not sufficiently centred over reference marker e Adjust the position of the camera using the blue camera movement arrow e Return to step 3 and follow the instructions from there onwards until there is a enough of the reference marker visible in the Marker detection window to cause the perimeter to turn green 8 When the perimeter of the Marker detection window is green the Spot Picker has detected a stable image of the reference marker 9 Below the Marker detection window there are two boxes which show the offset of the centre of the marker from the centre of the camera Marker X Off set Adjust Marker Y Off set 4 10 Press the Adjust button and the reference marker will be centered in the Marker detection window The marker is assumed to be centred when the values in both offset boxes are in the range 0 1 to 0 1 The procedure may need to be repeated until the offset values fall within the acceptable range Note Achieving 0 0 offset values is not required The software accounts the offset values so th

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