Home
PhastSystem - Bascom Palmer Eye Institute
Contents
1. PhastSystem User Manual 80 1320 15 Edition AK Operation 5 IMPORTANT When you program a method using temperature compensation you must program the process time t as the time required to process the step at 20 C regardless of the temperature you plan to run the step at Running the method Use the procedures for running development methods described at the beginning of the section Monitoring the method When a method is running the time shown on the display is based on the process time at 20 C and does not correspond to real time In other words the display time will elapse slower of faster than real time depending on the temperature of the solution in the chamber The following example will help clarify this A method is programmed with the following Ct curve DEV 7 Ct 5 30 40 50 C 0 5 1 3 2 0 2 6 The first step in the method is programmed as follows DEV7 01 IN 1 OUT 0 t 100min T 50 C That is the time for this process at 20 C is 10 minutes and it will be processed at 50 C When the step is running the display time will start at 0 0 minutes and count up to 10 minutes But the actual time taken to reach this display time 10 min may only be 4 minutes since the step is run at 50 C When the temperature of the solution in the development chamber is below 20 C the display time will elapse slower than real time When the temperature is above 20 C the display time will elapse faster than
2. Patent pending PhastSystem User Manual 80 1320 15 Edition AK 81 Installation 82 8 1 2 Spare parts This is a list of spare parts that might be required when following the maintenance outlined in chapter 7 A complete spare parts lists is contained in the service manual Designation Quantity Code no Separation and control unit Contact block cpl 1 18 3691 01 Contact pin 2 18 1661 01 Eccentric lever 1 18 1665 01 Sample applicator arm 1 18 1663 01 Separation bed cover 1 18 1671 01 Plunger adjustable 2 18 1019 67 Fuse 500 mA L 120 V model 5 19 8459 01 Fuse 250 mA L 220 V model 2 18 1000 68 PhastGel IEF gelcover 2 18 0083 01 PhastGel buffer strip holder 2 18 1668 01 PhastGel sample well stamp 1 18 0097 01 Development unit Gasket dev chamber 1 19 0048 01 Gasket 10 port valve 1 18 9482 01 Cap set 10 port valve 1 18 0072 01 Valve kit 10 port valve includes distributing plate and channel plate 1 18 1019 61 Tubing PVC 5 meters 1 19 0182 01 Tubing kit 10 port valve to chamber 1 18 0192 01 Fuse 175 mA L 120 model 2 18 1627 01 Fuse 80 mA L 220 v model 5 19 6236 01 Tube markers 0 9 5 of each 50 18 0180 01 Common items Fuse 800 mA L 220 V model 5 19 3085 01 Mains power cord 120 V 1 19 2447 01 Mains power cord 220 V 1 19 2448 01 Communication cable 1 19 6005 02 PhastSystem User Manual 80 1320 15 Edition AK Installation 8 2 Technical data 8 2 1 Separation and control unit A list of the
3. PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Sample applicators WA Fig 27 Inserting the applicators Starting the run 1 Press SEP start stop and enter the number of gels for this run NUMBER OF GELS 0 lt do gt Methods are programmed for 1 gel If you enter 2 gels here the current and power will be adjusted so that both gels run under the same conditions according to the programmed method See page 14 for details 2 Enter the number of the method you plan to run START SEP Method 0 0 lt do gt The method will start at the first step unless you renter a different step number after the period Once you enter the method number the method name will appear in parentheses to the right of the method number if you gave your method a name for example START SEP METHOD 4 0 SDS 10000 lt do gt 3 Press do to confirm Monitoring the run If the separation bedtemperature T is warmer or cooler than the programmed temperature T in the first step of the running method the display will show for example SEP 4 1 COOLING BED T 20 C T 18 C SEP 4 1 HEATING BED T 17 C T 18 C When T equals T the method will start and the running parameters will appear on the display for example SEP 4 1 400V 04 0mA 1 6W 18 C 0003A Vh First the accumulated volthours A Vh are shown that is the number of volthours that have elapsed since the beginning of the run By pressing SEP rea
4. Recycling This symbol indicates that the waste of electrical R and electronic equipment must not be disposed as unsorted municipal waste and must be mE Collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment Contents Contents 1 Introduction 2 Important safety information 21 Connection 16 themas SUD DIY cs cccecnceenmnctie ceuaeioenen 141 2 2 Safety arrangements cescesssocrevarnistcressssssanaseicivecenessiason sneteietenienemienneaniuetin 11 23 Safety Pe CORIO NS a n e ene eee KE Ee 12 3 Description of the system 3 1 The separation and control unit a eeeseeesssssscecssssssssneeessseecceecssssnseeeseeeeeeeesnnneesesess 13 32 Tie development Uni sniesrerscnsarennanu a aa 17 B S PhastGel media and SMES a srxcccccziearssssasticaidrerqerrssdiavactitversesnneriviniiienie Pil 34 Using the keyboarde ee 24 35 The PS NON ceca eee eee 25 4 Installation Gaim 0121 910 e E SD teEaIEa SP DTE UR neDeEE Fie oe 33 AZ IS AUS AIO a caceaasssssscinctvensconsscrasdieesiegneopasanizistebwssaaienneaueieiaaeremuaiiinisiealie 33 AS TUNGANE SUSTENO siisii omnes aetna ae 35 GA BEONE NN aces aces esercgeceiel ioaae oa Gee ere 35 5 Operation 5 1L Programming separation PFO CAUSE or y c cs rccsccecseesensseswrereshecsiessnesivereteercentce 37 52 Sample applicatio Messinia En EE 41 5 3 RUNNING TEF MEIO nnna n i ae 43 54 RUNNING electrophor
5. If valve leaks open and check that all parts are correctly mounted Note Ifthe valve still leaks you may have to replace the distributing and channel plates also Fixing plate Channel plate Distributing plate Gasket Distributor Pressure plate The 10 port valve wy Fig 39 The 10 port valve Start here The 10 port valve to chamber tube Fig 40 The 10 port valve to chamber tube PhastSystem User Manual 80 1320 15 Edition AK 79 7 Maintenance and trouble shooting 80 gt Replacing the valve to chamber tubing Disconnect the power cable Let the unit rest on the end opposite to the 10 port valve Remove the clamp at the valve end first and disconnect the tubing from the valve Remove the black cover plate for the tubing using a Philips screwdriver Shake the tubing to remove residual liquid Then remove the clamp at the chamber end Do not remove this clamp first residual liquid might then enter the unit Make sure that the new tubing rests in the recession when you put the cover back 7 3 Trouble shooting Help message reference You find enclosed here a reference for the help message that appear on the display when you press the help return key or at an alarm condition Trouble shooting guide You find enclosed here a trouble shooting guide that refers to the finished gel result 7 4 Recycling This symbol indicates that the waste of electrical and electronic equipment must not be dispose
6. or COPY DEV METHOD FROM 0 00 TO 0 00 lt do gt To copy a method enter the source method number at the cursor for example enter 1 COPY SET METHOD FROM 1 0 TO 0 0 lt do gt Press gt to move the cursor to the next field and then enter the destination method number tor example enter 2 COPY SEP METHOD FROM 1 0 TO 2 0 Press do to confirm Method one will be copied over to method two in this example To copy a method step enter the source method number and the method step for example enter 1 2 press 1 and 2 COPY SEP METHOD FROM 1 2 TO 0 0 lt do gt Press gt to move the cursor to the next field and enter the destination method and method step for example enter 3 4 COPY SET METHOD FROM 1 2 TO 3 4 lt do gt Press do to confirm Step 2 in method 1 will now be copied over to step 4 in method 3 DEL press to delete a method or method step Once you press this key the following command will appear on the display DELETE SEP METHOD 0 0 lt do gt or DELETE DEV METHOD 0 0 lt do gt PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 Enter the method number and press do to delete a whole method or enter the method number and step number to delete one step in the method For example press 1 and do to delete everything in method 1 DELETE SEP METHOD 1 0 lt do gt or press 1 2 and do to d
7. 2 Heat samples in SDS buffer 10 mM Tris HCl 1 0 mM EDTA pH 8 0 and 2 5 SDS 5 Bmercaptoethanol at 100 C for at least 5 min 1 Apply the sample under low current e g 1 0 mA Increase the separation time 2 Centrifuge or filter the sample 3 Use analytical grade SDS 99 pure to denature sample proteins 4 SDS precipitates upon freezing warm samples to 20 C before loading sample applicators 5 Dilute the sample 6 We recommend that sample applicators be used only once 1 Do not freeze at any time Frozen strips lead to proteins only running half the gel more blurry and a very low ending current 2 Always check the contact and gently bend the electrodes down a little if necessary 1 Avoid touching the buffer strips with anything when using sensitive staining methods Wear gloves Background smear on the gel PhastSystem User Manual 80 1320 15 Edition AK Development Coomassie staining Symptom Probable Cause Trouble Shooting Guide Solution s PhastGel Blue R 1 The coomassie stock solution or the final solution was too old and or unfiltered 2 Staining temperature is too low Stain particles on the gel surface 3 Cupric sulfate precipitates 4 Dirty tubing 1 The gel in the lower position of the gel holder was inserted gel side down The gel becomes splashed by incoming solutions 2 Old stain solutions or poorly filtered stain solutions A
8. 3 and do METHOD 3 NAME IEF 3 9 The name of the method will now appear on the display We will continue with method 3 as an example in the following instruction steps Programming sample application 10 Press step forward for the instruction SAMPLE APPL DOWN AT 3 0 0000 Vh 11 Enter the step number when the sample applicator is to be lowered onto the gel for example enter step 1 SAMPLE APPL DOWN AT 3 1 0000 Vh 12 Press gt and enter the time volthours in the current step where the sample will be applied to gel step 1 in this example For example 75 volthours SAMPLE APPL DOWN AT 3 1 0075 Vh 13 Press step forward to program the second sample application instruction Enter the step number can be the same as for applicator down press gt and enter the volthours that will elapse before the sample applicator is raised trom the gel for example SAMPLE APPL UP AT 3 1 0150Vh Programming the extra alarm 14 Press step forward for the extra alarm instruction Enter the step number press gt and enter the number of volthours that will elapse in this step before the extra alarm sounds for example EXTRA ALARM TO SOUND AT 3 1 0073 Vh The extra alarm can be programmed to sound anytime during the run for example to let you know when it s time for sample application so that you can pause the run if you need more time to load the sample applicators Note An alarm s
9. PhastGel Blue R is a Coomassie R 350 stain stamped into convenient tablet form The tablets are first dissolved in water Methanol is then added and this solution is filtered and stored as a stock solution Before use acetic acid is added The final solution is only stable for about one day One pack of PhastGel Blue R contains 40 tablets Each tablet makes 400 ml of 0 1 stain solution Instructions for storage and use are included with every pack Optimized development methods using PhastGel Blue R are described in chapter 9 Development technique file No 200 and 201 PhastSystem User Manual 80 1320 15 Edition AK 23 3 Description of the system 24 PhastGel silver kit Silver staining is traditionally a complex method with several steps that are both time and temperature sensitive With the automated development in PhastSystem even complex methods have become easy to manage reproducibly With PhastGel silver kit all the solutions that can be critical are conveniently packed in ready to use bottles The user only has to provide ethanol acetic acid trichloroacetic acid glycerol tris HCl and water all normally available in most laboratories 3 4 Using the keyboard The aim of this section is to prepare you for programming editing and running separation and development methods Before you begin to program or run a method you should know what happens when you turn on the system know what the LEDs are form and befamiliar with the
10. The gels can be restained even if they are dry Run the stain and destain steps again PhastSystem User Manual 80 1320 15 Edition AK Trouble Shooting Guide Symptom Probable Cause Solution s No bands but blue background Small cracks and rough uneven surface afer drying Solution bottles were incorrectly connected to the ports according to the programmed method or the wrong method was started Finger proteins from handling the gel without using forceps or gloves This usually occurs when gels are dried too fast e g the air stream from the hair dryer is too hot or too high Always label bottles with their corresponding port number Label tubes with their corresponding port numbers using the yellow tubing markers Check the program against the bottle arrangement Use the plastic tab from the gel backing to handle the gels Use forceps Use the lowest heat setting on the hair dryer PhastSystem User Manual 80 1320 15 Edition AK PhastGel IEF media Symptom Probable Cause Trouble Shooting Guide Solution s Dark area across the gel Dark background Dark uneven background staining PhastSystem User Manual 80 1320 15 Edition AK No or too little copper sulphate in the staining solution Old fix and wash solutions Old fix and wash solutions the first two solutions This is usually visible only after the gel is dry The coomassie concentr
11. sample wells well volume PhastGel sample applicator 6 4 sample wells well volume Patent pending 5 0 T 3 C 7 5 T 2 C 7 5 T 2 C 30 ethylene glycol 20 T 2 C 30 ethylene glycol 41x10x6mm approx 2 5 ml 3 Agarose IEF 0 88 M L Alanine 0 25 M Tris 0 20 M Tricine 0 20 M Tris 0 55 SDS 8 8 8 1 4 8 C Do not freeze 12 approx 0 3 ul of sample 8 approx 0 5 ul of sample 8 approx 1 ul of sample 6 approx 4 ul of sample PhastSystem User Manual 80 1320 15 Edition AK www gehealthcare com GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden PhastSystem PhastGel and Pharmalyte are trademarks of GE Healthcare companies GE imagination at work and GE monogram are trademarks of General Electric Company Parafilm is a trademark of American National Can All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information 2006 General Electric Company All rights reserved GE Hea GE Hea Bjorkga GE Hea tl tl tl hcare Bio Sciences AB a General Electric Com
12. 1320 15 Edition AK 43 5 Operation 5 Wipe off the separation bed with a moist lint free cloth to remove dust or particles It is also advisable to wipe off the electrodes gently with a cloth that does not leave dust or other particles Note In order to obtain the best results possible we recommend frequent cleaning of the electrodes Even minor amounts of deposited impurities have been shown to sometimes affect the resolution and band pattern See also Maintenance chapter 7 Eccentric lever in lower position Fitting the IEF gel cover Fig 18 Fitting the IEF gel cover Positioning the gels 1 Place a drop of water or insulating fluid approximately 60 75 ul onto the middle of the gel area s outlined by the red lines on the separation bed 2 Take one or two gels from the refrigerator Use a pair of scissors to cut the package along three sides Make sure that the thin plastic film on the gel does not stick to the package inside Remove the gel from its package with a pair of forceps use the plastic tab of the gel backing as a handle The thin plastic film on the gel surface protects the gel from contaminants and from drying and should be left on for now 3 Use a waterproof pen to mark the underside of the gel for identification You might have to wipe the back of the gel first 44 PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Fig 19 Bending the tab up 4 Place the gel on a hard surface
13. 2 4 PhastGel Separation media ANA ACCESSOSICS sssssssssssssssssssssssssssecssssssssssssssessssssssssssseeeees 85 6 PhastSystem User Manual 80 1320 15 Edition AK Introduction 1 1 Introduction PhastSystem consists of a separation and control unit a development unit high performance PhastGel separation media accessories and a technical support package These components work together to form a system for fast high resolution and reproducible electrophoresis With PhastSystem isoelectric focusing is as easy to perform as gel electrophoresis Coomassie staining is as easy as silver staining The schematic diagram below illustrates the steps involved in producing a finished electrophoresis gel using PhastSystem with PhastGel separation media Flow diagram for PhastSystem Separation and control unit Development unit Place 1 or 2 gels on Place gells in the separation bed 3 min ad development chamber 1min Load PhastGel 3 min Select a programmed sample applicator s development method and press the start button Select a programmed when method stops 30 90 min separation method and press the start button remove the gells and analyze the results when alarm sounds 20 45 min Total time 32 92 min remove the gel s Total time 26 50 min or J gt Specific detection via conventional methods e g Zymograms auto radiography blotting T
14. 75 Vh during step 1 During this 75 Vh period the sample applicators can be loaded An alarm will sound after 73 Vh as a warning that sample application will occur in 2 Vh After 150 Vh in step 1 the sample applicators will be raised from the gels thus sample application will occur for 75 Vh When the applicators are raised step 1 will continue for another 350 Vh After 500 Vh an alarm will sound to mark the end of method but step 1 will continue to run until the method is stopped by pressing SEP start stop Important Running methods will not stop until you press SEP start stop When a running method reaches an empty not programmed method step an alarm will sound to mark the end of the method but the method will continue to run with the same conditions as those programmed in the step This is to prevent band diffusion should the method end when you are beyond hearing distance of the alarm To reduce the risk of gradient drift or SDS denatured proteins migrating off the gel you can do one or both of the following PhastSystem User Manual 80 1320 15 Edition AK Operation 5 1 Program the last step as a low voltage 100 V for SDS PAGE or 1000 V for IEF step of 0 volthours The alarm will sound immediately once this step is reached but the method will continue to run at low voltage until you press SEP start stop 2 Program the extra alarm to sound before the last step is finished This will inform you
15. Manual 80 1320 15 Edition AK Operation 5 The temperature compensation factor for 50 C is 2 6 that is the process will reach completion 2 6 times faster than it would at 20 C When the solutions for this step are pre heated to 50 C 4 the process ends exactly 2 6 times faster than when the process is run at 20 C 12 0 min 2 6 4 6 min Estimating Ct factors The Ct curve is an average for the entire method and it can be estimated in a number of different ways The following is a general procedure that you can use and modify to estimate the Ct curve for your method 1 Leaving all Ct factors set to 1 0 run your method all steps at 5 20 30 40 and 50 C using solutions pre cooled or preheated to these temperatures Use at least two different times at each temperature 2 Change the process time equally for each step in the method For example if you halve the process time in the first step for the run at 40 C halve all the steps in the method If your method contains a step that cannot he run at high temperatures increase the temperature of this step along with the other steps until you reach the maximum allowable or optimum temperature for that step Then leave the step at that temperature and continue increasing the temperature for the other steps 3 Plot the staining intensity versus time for each temperature The staining intensity can be measured visually or with a densitometer Use an arbitrary scale
16. and control unit the development unit and PhastGel media and chemicals After you have read this chapter you will know what the components look like how they function and how they worktogether to form a system for fast electrophoresis PhastSystem 3 1 The separation and control unit The separation and control unit is the heart of PhastSystem because it contains the microprocessor which controls and monitors both separation and development processes according to programmed methods Methods are programmed using the keyboard The LCD display shows the method steps during programming When separation and development methods are started the display shows the actual running conditions so you can monitor the progress of the methods The separation and control unit also contains the separation compartment and the power supply The microprocessor separation compartment and power supply are described below The keyboard is described in a following chapter Microprocessor The microprocessor in the separation and control unit controls and regulates all parameters during separation and development runs Methods are programmed using the keyboard and stored in a semiconductor memory This memory is guarded by a battery so that methods are not lost when the system is turned off or if mains power fails Every time the system is turned on the microprocessor does a diagnostic test to make sure everything functions properly If an error is detected a me
17. backward cursor help Ki return cursor Fig 13 Programming keys 26 PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 Programming keys The keys in the two centre blocks on the keyboard are used for programming and editing programmed methods The help return key and the do key are used for both programming and run control Below the function of each programming key is described following the key name SEP method file press to enter the separation method file DEV method file press to enter the development method file Within a method step the position for an entry is marked out by an underscore cursor on the display gt press to move to the next field of entry within a method step lt press to move to the previous field of entry within a method step You can move the cursor rapidly through a method step by depressing these keys longer than one second These keys also serve as stepping keys for selecting characters when naming a method step forward f step forward press to move to the next step in a method step backward ee step backward press to move to the previous step in a method You can move quickly through a method by depressing these keys longer than one second These keys also serve to select a method number when naming a method name name method press to assign a name maximum of 10 characters to a m
18. but press DEV pause continue as soon as EMPTYING PO appears on the display When the 10 port valve is in position PO the channel groove in the channel plate is always pointing to 12 o clock 2 Disconnect the development unit mains power cable 3 Raise the valve end of the unit about 300 Do not stand the unit on end or residual liquid may enter delicate parts in the unit 4 Unscrew the pressure plate taking a few turns at a time on each screw 5 Remove the distributor and distributing plate see figure Closing the valve 6 Without touching the surface of the distributing plate place gasket against the smooth side notch in gasket against notch in distributing plate 7 Place the two parts into distributor 8 Reassemble valve Remember channel groove in channel plate should be pointing to 12 o clock The valve will not connect ports as programmed if the channel plate is turned by 180 9 Make sure the u shaped metal piece is sitting under the valve and that the hole for the screw is to the right Insert the screws in the pressure plate Screw in the left and right screws first a few turns then the bottom screw and finally the top screw Tighten the left and right screws and the top and bottom screws just until there is resistance PhastSystem User Manual 80 1320 15 Edition AK Maintenance and trouble shooting 7 10 Re connect mains power cable Press DEV pause continue and then DEV start stop
19. check that they are straight Hold the assembly up to eye level to check this When you carry out an electrophoresis run again confirm that there is good and even contact between the electrodes and the buffer strips PhastSystem User Manual 80 1320 15 Edition AK Maintenance and trouble shooting 7 Contact block Contact pin Fig 37 The electrode assembly Replacing the contact pieces The contact blocks and pins should be replace when damaged When you have pulled out the applicator arm and electrode assembly unit lay it down on a table and pull out the contact blocks Replace the blocks and fit the eccentric levers into place It might help to move the lever a little back and forth until it slips into place When you replace one contact pin always replace the other Replacing the separation bed cover The separation bed cover should be replaced if the surface has been damaged by burns or deep scratches Remove the damaged cover by lifting one of the edges up with a scalpel and peeling the cover off If there is any glue left on the cooling plate remove it by moistening a piece of cloth in an adhesive solvent e g terpentine or ligroin and tuck it down into the recess The cloth must be drip free solvent might otherwise dissolve the insulation below the separation bed Rub gently or leave it on for about one hour to dissolve the old adhesive Wipe away the old adhesive and clean the bed thoroughly the new gel bed cover m
20. continue 5 2 Sample application Since the procedure for loading sample applicators is the same for all media the procedure is described separately here Different separation techniques require different sample preparation Guidelines for sample preparation salt and sample concentrations are given in the Separation technique files Loading sample applicators Samples are applied to gels with PhastGel sample applicators The choice of sample applicator will depend on the number and volume of the samples you want to apply For example the PhastGel sample applicator 8 0 5 will apply eight samples each approximately 0 5 ul to the gel The PhastGel sample applicator TC is a special applicator for electrophoretic titration curve analysis PhastSystem User Manual 80 1320 15 Edition AK 41 5 Operation 42 The PhastGel sample well stamp forms correctly spaced depressions in strips of Parafilm from which the desired size of sample applicator may be loaded Samples are pipetted into the depressions and are drawn up into the applicator capillaries by capillary action The actual volume of the sample drawn up will depend on its surface tension the higher the surface tension the larger the volume held in the applicator capillary Therefore for quantitative purposes the applicator capillaries should be filled with an exact volume of sample using a syringe Using the sample well stamp 1 Place the sample well stamp onto a table wi
21. equals the programmed time for the step the chamber empties and the out port number PI is shown on the display for example DEV 7 01 t 10 5min T 50 C EMPTYING P1 After the chamber is emptied the in port tube is cleared of solution by compressed air The display will show for example DEV 7 01 t 0 0 min T 26 C CLEANING TUBE PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Subsequent steps The subsequent steps will be carried out in the same manner except there is no initial emptying step When the method reaches an empty step not programmed the method ends and the display will show for example DEV t 0 0 min METHOD 7 DONE Interrupting the run If any thing goes wrong while the method is running you can stop the run temporarily or terminate it To stop the run temporarily Press DEV pause continue The DEV ON LED will blink to show the method is paused and an alarm will sound at 20 second intervals until the run is continued Press DEV pause continue when you are ready to continue The DEV ON LED will show a steady light again To terminate the run Press DEV start stop The display will prompt you to confirm PRESS do TO END METHOD When you press do the development chamber empties and the in port tube is cleared The display will show for example DEV 7 8 t 5 0 min T 45 C ENDING METHOD When the chamber is empty the display will show for example DEV t
22. for visual detection 4 Draw a line parallel to the x axis across each curve at an appropriate O D or staining intensity and read the time required for the reaction to reach this value at different temperatures An example is shown in Figure 32 Comparing the process speed at different temperatures 50 C yas o 5 40 C 30 C agec 5 C Band intensity O D E 5 10 15 20 25 30 35 60 Time min Fig 32 Comparing the rate at a process at 20 C with the rate of that process at 5 30 40 and 50 C PhastSystem User Manual 80 1320 15 Edition AK 63 5 Operation 64 5 Use the process time at 20 C as the reference time and estimate the Ct factors for 5 30 40 and 50 C divide the reference time 130 minutes in Fig 3 by the time required for the respective runs For this example shown in Figure 3 the Ct factors are e for 5 C 30 min 60 min 0 5 e for 20 C 30 min 30 min 1 0 e for 30 C 30 min 23 min 1 3 e for 40 C 30 min 15 min 2 0 e for 50 C 30 min 11min 2 6 The Ct factor for 20 C is always 1 0 6 Plot the Ct factors against their respective temperatures to obtain the Ct curve for your method as shown in Fig 1 An alternative method for estimating Ct factors is to optimize the development method at 50 C or some other temperature without using temperature compensation Then calculate the time that would be required for each method step at 20 C using the general
23. instructions for safe operation and maintenance of the system 2 1 Connection to the mains supply Voltage selector setting The instruments are available in two versions one for 220 230 240 V AC referred to here as the 220 V model and one for 100 120 V AC referred to here as the 120 V model As a safety precaution check the code number and voltage printed on the backpanels to ensure you have the correct model for your local electricity supply Code number 18 1018 23 Separation and control unit 120 V model Development unit 120 V model Code number 18 1018 24 Separation and control unit 220 V model Development unit 220 V model Set the voltage selectors on the rear panels of the separation and control unit and development unit according to your local electricity supply To do this e Check the voltage range of the mains electricity supply e Set the voltage selector to the appropriate setting according to the table below Voltage range Voltage selector setting For 120 V model instruments 90 110 100 108 132 120 For 220 V model instruments 198 242 220 230 216 264 240 Important Always disconnect the mains power cords when servicing the system 2 2 Safety arrangements The operator is protected against high voltage by the separation compartment lid when an electrophoresis is in progress If the lid is opened during a run the high voltage supply switches off automatically to eliminate electr
24. is divided into two major parts separation problems and development problems Each part is subdivided into problem common to both PhastGel IEF homogenous and gradient media and problems specific to each of these media when applicable The development section includes trouble shooting guidelines for both coomassie and silver staining Note This guide is specifically concerned with the problems that might occur with the techniques presented in the technique files e g coomassie staining with Phastgel Blue R These guidelines may or may not pertain to other methods using different reagents Separation Symptom Probable Cause Solution s 1 The plastic film on the gel 1 Remember to remove the film surface was not removed before starting a run 2 No electrode contact with 2 Check the electrodes to make No bands the gel sure they are even Gently on the gel bend them down a little Always check the electrode contact with gel or buffer strips 3 The sample was not 3 Check that the sample applied to the gel applicators gel cover and buffer strip holder are positioned correctly 1 Excess water on the bed 1 Remove excess water after surrounds and floods the gel positioning gels onto the separation bed Use only about 60 75 ul under each gel Increase the temperature of the bed if the humidity in your Disturbances at one lab is too high or both of the edges gt Samples were applied too 2 Make sure the ge
25. native buffer strips Extra bands with native PAGE The native buffer strips were used instead of the SDS buffer strips Long streaks without any bands for SDS PAGE 1 Strips had uneven contact with the gel 2 The gel was not positioned properly so that proteins migrate too close to one edge Be sure to use the correct buffer strips Be sure to use the correct buffer strips 1 Gently press down along the buffer strips to ensure good contact with the gel Wear gloves or use a smooth object to do this 2 Be sure the gel is positioned within the vertical red lines in the separation bed Curved bands on one or both sides of the gel PhastSystem User Manual 80 1320 15 Edition AK Trouble Shooting Guide Symptom Probable Cause Solution s Loss of protein bands appearance of extra bands Streaking Proteines only runing half the gel 1 Proteolysis of sample proteins 2 Incomplete protein dissociation with SDS PAGE 1 Poorly soluble proteins 2 Particulates in the sample 3 Impure SDS in the sample 4 Samples stored frozen with SDS and not warmed prior to electrophoresis 5 Sample overloading 6 Dirty sample applicator 1 Frozen buffer strips 2 Not good enough contact between buffer strips and electrodes 1 Touching buffer strips with fingers 1 Prepare samples at low temperature Try adding protease inhibitors
26. occur Depending on the magnitude of the temperature drift results may or may not be affected Therefore use this graph as a guide not as a rule When choosing a separation bed temperature the humidity in the room must also be taken into account or excessive condensation might affect results 9 w 30 For 2 gels je T 7W 25 ou 2 E 20 o 4W Lv Q S i 2 lt lt O lt A 5 7W e won 4W 0 er oe 10 15 20 25 30 35 40 Room temperature C Fig 5 Cooling capacity vs ambient temperature The lowest separation bed temperature achievable with deviations less than 1 C with ambient temperature up to 40 C for two gels run at 4 W and 7 W 3 2 The development unit The visible parts of the development unit are a stainless steel chamber with a heating foil a rotating gel holder for one or two gels a temperature and level sensor on the underside of the lid and ten ports through which the development chamber can be filled and emptied Ports labelled 1 9 are used to connect development solutions to the development chamber The port labelled O is reserved for waste that is solutions only exit through this port The gel holder liquid level sensor and temperature sensor are mounted in the lid of the development chamber and protrude into the chamber when the lid is closed Inside the unit there is a pneumatic pump for filling and emptying the chamber a 10 port valve for the selection of
27. ports and a 3 port valve for the selection of pump functions i e creating vacuum or pressure in the chamber The pneumatic pump is connected to an opening in the lid of the chamber A gasket in the lid makes the chamber airtight when the lid is closed By creating a vacuum in the chamber liquid is drawn in through a hole in the bottom of the chamber Similarly by creating excess pressure in the chamber liquid is pushed out through the same hole in the bottom PhastSystem User Manual 80 1320 15 Edition AK 17 3 Description of the system 18 Development methods Nine development methods are available for programming For each method you can program up to 20 steps For each step the in port for filling the out port for emptying the duration of the step in minutes and the temperature for processing the gel the chamber can heat solutions up to 50 C is programmed As programming options each development method can have a temperature compensation curve and an extra alarm to sound at a set time during the run More information about temperature compensation is given in Development procedures section 5 3 Once the bottles of development solution are connected to the ports by the PVC tubing the gels are inserted into the gel holder the lid is closed the start button is pressed and the rest is automatic The method ends when it reaches an empty unprogrammed step Level sensor N Bz Temperature sensor j j S Gel ho
28. range Number of prot per Unused Reconst kit vials kit vial HMW kit SDS 18 500 10 250 5 4 C 20 C 330 000 Native 67 000 670 000 HMW SDS SDS 53 000 10 200 5 20 C 20 C kit 212 000 LMW kit SDS 14 400 10 600 6 4 C 20 C 94 000 PMW kit SDS 2 512 di 2000 5 4 C 20 C 16 949 1 Designed for use with SDS electrophoresis only Procedures for MW measurement Each MW calibration kit is supplied with complete instructions for use Follow these instructions but dilute the vials with 200 ul of buffer for Coomassie staining and with 3 ml of buffer for silver staining instead of 100 pl as suggested in the instructions A condensed version of the instructions is given below to illustrate the simplicity of the method 1 Dissolve the calibration kit proteins in 200 ul for Coomassie staining or 3 ml for silver staining of suitable buffer for electrophoresis For native PAGE reconstitute one HMW calibration kit vial in distilled water For SDS PAGE reconstitute one HMW or LMW or both vial in 10 mM Tris HCl pH 8 0 1 0 mM EDTA with 2 5 SDS and 5 0 B mercaptoethanol Mix by gently swirling Heat this mixture at 100 C for 5 10 minutes Reconstituted denatured kit proteins can be stored frozen at 20 C 1 2 A 5 Prepare the sample proteins in the appropriate buffer as given above Carry out electrophoresis according to the method given for native or SDS PAGE in the Separation Technique Fil
29. rule that the process rate will double for every 20 C rise in temperature Program these values into the method but leave the temperatures the same those obtained when optimizing the method Program the Ct curve and run the method to test the curve The curve can then be adjusted and tried again until you are satisfied it fits the method Programming the method Program your development method using the procedure given at the beginning of this chapter but program the process time as the time required for the step if it were processed at 20 C Instructions for programming the Ct curve are given below Program the Ct Curve as follows 1 Press step forward until the Ct curve instruction appears on the display for example DEV 7 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 2 To program the Ct factor for 5 C you must first erase the default value 1 0 Press CE DEV 7 Ct 5 30 40 50 C 0 0 1 0 1 0 1 0 Then enter the Ct factor for example enter 0 5 DEV 7 Ct 5 30 40 50 C 0 5 1 0 1 0 1 0 3 Press gt to move the cursor to the next position press CE and enter the Ct factor tor 30 C for example 1 3 DEV 7 Ct 5 30 40 50 C 0 5 1 3 1 0 1 0 4 Press CE and enter the Ct factor for 40 C for example 2 0 DEV 7 Ct 5 30 40 50 C 0 5 1 3 2 0 1 0 5 Press CE and enter the Ct factor for 50 C for example 2 6 DEV 7 Ct 5 30 40 50 C 0 5 1 3 2 0 2 6
30. technical data for PhastSystem instruments is given below Dimensions 460 x 300 x 138 mm W x L x H Weight 6 2 kg Keyboard 31 tactile keys Display 40 digit alphanumeric liquid crystal display LEDs 4 LEDs for status information Alarm An audible alarm sounds at the end of separation methods Capacity 1 or 2 gels Programs Separation 9 methods available for programming Development Programmable parameters Separation Development Internal power supply Circuitry protection Voltage range Error Current range Error Power range Error Internal Vh integrator range Battery back up memory Separation compartment Electrodes number material width spacing Cooling heating Temperature Sample application Safety precautions PhastSystem User Manual 80 1320 15 Edition AK each method contains 9 steps 9 methods available for programming each method contains 9 steps Volt current power temperature duration in volthours sample application and an extra alarm Inlet port outlet port duration in minutes temperature temperature compensation curve and an alarm Short circuit protection 10 2000 VDC lt 3 of actual value 5 V for 10 2000 VDC 0 1 50 0 mA lt 2 of actual value 0 2 mA for 0 1 5 0 mA 0 1 7 0 W lt 6 of actual value 0 3 W for 0 1 1 0 W Integrates volts with time 1 9999 volthours step Lithium battery shelf life 10 years 1 cathode and 2 anodes gel Platinized tit
31. that the run is almost finished Editing a method To edit a programmed method press SEP method file and select the method you wish to edit You can select the step you want to change by entering the step number after the period for example to edit step 3 in method 1 press 1 3 and then do GET SEP METHOD 1 3 lt do gt SEP 1 3 0300V 07 0mA 2 0W 15 C 0010Vh Alternatively start from the beginning of the method and press stepforward until the step you want to edit appears on the display Use the gt and lt 4 keys to move to the field you want to change Once the cursor rests under the entry you want changed press CE SEP 1 3 0000V 07 0mA 2 0W 15 C 0010Vh Then enter the new value To insert a step or copy or delete a method or method step see Using the keyboard where these keys are described To start a run see p 44 or 51 Editing a running method To edit a running method you must first press SEP pause continue unless you only want to program or change the extra alarm Then select the method in the SEP method file To change an entry in a running method follow the directions above for editing a programmed method You can delete or insert a step in a running method only if the deleted or inserted step follows the step in progress when you paused the run Running methods cannot be deleted Do not forget to continue the run when you finish editing your method press SEP pause
32. the gels After a programmed interval the applicator arm is raised again An alarm will sound to mark the end of the last step in a running method But methods will continue to run with the same running conditions as the last step until the method is stopped by pressing the stop key This is to prevent band diffusion in case you miss the alarm Power supply The power supply can be programmed to function in three modes constant current constant voltage or constant power by setting limits on these parameters The microprocessor automatically adjusts the parameters during each step in a separation method PhastSystem User Manual 80 1320 15 Edition AK 15 3 Description of the system 16 For maximum reproducibility the duration of each method step and the time for sample application is measured in volthours Volthours indicate the extent of protein migration in the gel since electrophoretic mobility is proportional to the applied voltage and the time that this voltage is applied Since the voltage change continually the unit is equipped with a volthour integrator which integrates volts with time The extra alarm is also programmed in volthours For more information about volthours and volthour integration see reference 1 1 Isoelectric Focusing In Gel Electrophoresis and Isoelectric focusing of Proteins Allen R C Saravis C A Maurer H R editors Walter de Gruyter Berlin and New York 1984 p 76 Allen R C Cooling c
33. the locking screw in case you should ever need to ship the unit Leaving the screw in place willmake the unit noisier but will not affect the operation Unpacking the electrodes Carefully remove the plastic packing material from the electrode unit in the separation compartment of the separation and control unit Check that the electrodes are straight 4 2 Cable connections Voltage selector setting PhastSystem instruments are available in two versions for 220 240 V AC and for 110 120 V AC electricity supplies Check that the instruments have the correct voltage and code number printed on their back panel 220 240 V 18 1018 24 Separation Control and Development Units 18 1200 10 Separation Control Unit 110 120 V 18 1018 23 Separation Control and Developments Units 18 1200 00 Separation Control Unit PhastSystem User Manual 80 1320 15 Edition AK 33 4 34 Installation Set the voltage selectors on the rear panel of the separation and control unit and development unit according to your local electricity supply 110 120 V or 220 230 240 V To do this e Check the voltage range of the mains electricity supply e Set the voltage selector to the appropriate setting according to the table below Voltage range Voltage selector setting For 120 V model instruments 90 110 100 108 132 120 For 220 V model instruments 198 242 220 230 216 264 240 Fuses Each unit has two fuses Check that the fuses are correctl
34. 000 100 000 40 000 20000 4 10 000 0 2 0 4 0 6 0 8 Fig 36 The calibration curve established using the LMW calibration kit for the gel shown in Fig 35 The gel was projected onto a 25 x 25 cm format for measuring band distances The proteins starting from the cathode and their corresponding molecular weights are phosphorylase b 94 000 albumin 67 000 ovalbumin 43 000 carbonic anhydrase 30 000 trypsin inhibitor 20 100 a lactalbumin 14 400 PhastSystem User Manual 80 1320 15 Edition AK Maintenance and trouble shooting 7 7 Maintenance and trouble shooting In this chapter instructions are given for the maintenance of the instruments parts that your service department can easily and quickly perform This chapter begins with instructions concerning both instruments changing the fuses and calibrating temperature sensors Then the chapter is divided into two parts maintenance instructions for the separation and control unit and for the development unit IMPORTANT Always disconnect the power when service the instruments At the end of the chapter you will find a reference for the help messages that appear on the display when you press the help return key or at an alarm condition You will also find a trouble shooting guide that refers to the finished gel result Fuses To remove a fuse to check if it has blown press the fuse holder in with a screwdriver and turn in counterclockwise If the fuse blows a
35. 4 gt PROGRAM AN EXTRA ALARM Although an alarm sounds automatically at the end of a separation method you may want to program an extra alarm to sound at any time during a run Program the alarm as you would for sample application see help messages 10 13 above With the cursor in any of the V mA Q or C fields in a method step SEP 1 3 0000V 00 0MA 0 0W 00 C 0000Vh 15 gt MAX U 2000V 25 0mA P 7 0W T 70 C These are the maximum values you can program for a separation method Since methods are programmed for one gel these values are the maximum running values for one gel For two gels the maximum running values will be one half the maximum current and power i e 12 5mA and 3 5 W See help message 17 below SEP 1 3 0000V 00 0mA 0 0W 00 C 0000Vh 16 gt MAX VOLTHOURS IN A STEP 9999Vh When starting a separation run NUMBER OF GELS 0 lt do gt 17 gt IF TWO GELS AND P ARE COMPENSATED When you run two gels the current and the power will be compensated so that both gels run under identical conditions For example if a method programmed with 12 0 mA and P 2 0 W is run using two gels each gel will receive 12 0 mA and 2 0 W See page 37 of the manual START SEP METHOD 1 0 lt do gt 18 gt PROCESS WILL START AT THIS STEP If you do not enter a step number the method will start at step 1 automatically PhastSystem User Manual 80 1320 15 Edition AK Help Message Reference Development field messages Press hel
36. 5 0 min METHOD 7 DONE 5 7 Cleaning method Before using the development unit for the first time you should run a cleaning method to rinse the development chamber and tubing from dust accumulated during storage and shipment Also before running a sensitive staining technique such as silver staining you may want to clean the chamber and tubing thoroughly Use the instructions below to program and run a cleaning method 1 Switch on the system 2 Press DEV method file Method 9 will be used in this and the following examples 3 Press name method and press step forward until you reach method 9 DEV 9 L A Method 9 will be called CLEANING in this example 4 Press P until C appears in the parentheses and press do to enter it into the name field DEV 9 C C 5 Follow step 4 to enter the rest of the characters 6 Press DEV method file again PhastSystem User Manual 80 1320 15 Edition AK 59 5 Operation 10 Press 9 and do Press step forward Leave these values set to 1 0 DEV 9 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 Press step forward Leave the alarm instruction blank EXTRA ALARM TO SOUND AT 9 00 t 00 0min Press step forward for the first method step In steps 1 through 9 program the in port IN 0 to correspond to the step number for example IN 1 IN 2 IN 3 for steps 1 2 and 3 respectively Leave the out port and the temperature T set to ze
37. 6 of the manual DEV start stop 5 gt DEV WILL EMPTY AUTOMATICALLY IF lt do gt If you press do the method will end after the chamber empties SEP standby temp 6 gt TEMP WHEN SEP METHOD IS NOT RUNNING The separation bed will be cooled or heated to the temperature you program You must press do to activate the standby temperature you entered See page 29 of the manual name method 7 gt TO NAME A METHOD PRESS lt cursor gt amp lt do gt Once you press name method you must press step forward to display the method number you want to name Then you must press gt or lt 4 to call up a character and do to enter it into the name field See page 25 of tne manual PhastSystem User Manual 80 1320 15 Edition AK 1 Help Message Reference Separation field messages Press help return to display information a field marked out with the cursor When programming a separation method GET SET METHOD 0 0 FREE 123456789 8 gt MAxX NUMBER OF SEP METHODS IS 9 GET SEP METHOD 0 0 FREE 123456789 8 gt MAX NUMBER OF SEP METHODS STEPS IS 9 SAMPLE APPL DOWN AT 1 0 0000 Vh 10 gt ENTER STEP NUMBER FOR SAMPLE DOWN SAMPLE APPL DOWN AT 1 0 0000 Vh 11 gt VOLTHOURS ELAPSED BEFORE SAMPLE DOWN SAMPLE APPL UP AT 1 0 0000 Vh 12 gt ENTER STEP NUMBER FOR SAMPLE UP SAMPLE APPL UP AT 1 0 0000 Vh 13 gt VOLTHOURS ELAPSED BEFORE SAMPLE UP EXTRA ALARM TO SOUND AT 1 0 0000 Vh 1
38. EV METHOD 7 00 COOM IEF lt do gt The method always starts at the first step unless you enter a different step number after the period 3 Press do to confirm During the course of the run you may at any time start a separation run or program another separation or development method Press DEV real condition to display the progress of method Monitoring the progress The display will keep you informed about the progress of the method and the event that is taking place The sequence of events during a development step is described below First the development chamber is emptied of eventual residual liquid through port O PO to waste This is a precautionary step it is not programmable DEV 7 01 t 0 0 min T 22 C EMPTYING PO Next the chamber will be filled with liquid through the programmed in port for example through port 1 P1 DEV 7 01 t 0 0 min T 22 C FILLING P1 t takes approximately 15 seconds to fill the chamber When the chamber is full the solution is heated to the programmed temperature and the gels are rotated in the solution until the end of the step that is the gel is being processed DEV 7 01 t 0 5 min T 28 C PROCESSING P1 The in port tube number PI will remain on the display until the chamber empties The time t shown on the display starts at zero and counts up to the time programmed for the step The temperature T is the actual temperature of the solution When the processing time
39. GE Healthcare PhastSystem Automated electrophoresis User Manual Important user information All users must read this entire manual to fully understand the safe use of PhastSystem WARNING be followed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood h The WARNING sign highlights instructions that must CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product CE Certifying This product meets the requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request 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 GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual 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
40. ITED IS SEP OFF When trying to pause a separation run not started 106 gt INSERT ALLOWED ONLY WITHIN METHOD When trying to insert before entering a method in the program mode 107 gt TO EDIT A RUNNING METHOD DO lt pause gt When trying to edit a running method without pressing SEP pause continue or DEV pause continue first 108 gt source method does not exist When trying to copy a free not programmed method 109 gt METHOD STEP 0 DOES NOT EXIST When trying to call up step 0 steps are numbered 1 to 9 in separation methods and 1 to 20 in development methods 110 gt INSERT ALLOWED ONLY AT METHOD STEPS When trying to insert at an applicator or alarm instruction in a separation method or at a Ct or alarm instruction in a development method 111 gt NO FREE STEPS AFTER PRESENT When trying to insert a step in a method when all subsequent steps are programmed You may insert if you first delete one of the subsequent steps 112 gt DESTINATION METHOD OCCUPIED When trying to copy a method to a programmed method 113 gt COPY FROM METHOD TO STEP PROHIBITED When trying to copy a whole method into one step 114 gt SOURCE METHOD STEP DOES NOT EXIST When trying to copy a step that is not programmed 115 gt DESTINATION METHOD STEP OCCUPIED When trying to copy to a step that is already programmed 116 gt DELETE OF RUNNING METHOD PROHIBITED When trying to delete a running method You must stop the method first before you delete
41. Pharmalyte concentration pH gradient PhastGel gradient media Dimensions Stacking zone composition Length Gradient zone composition Length Buffer system pH PhastGel homogeneous media Dimensions Stacking zone Separation zone Buffer system pH PhastGel homogeneous 20 Stacking zone Separation zone PhastGel homogeneous 12 5 Stacking zone Separation zone PhastSystem User Manual 80 1320 15 Edition AK polyacrylamide polyester 0 175 mm thick 4 8 C Do not freeze 43 x 50 x 0 35 mm 5 T 3 C 37mm approx 22 umol ml gel pH unit linear 43 x 50 x 0 45 mm 6 T 3 C 13mm PhastGel gradient 10 15 10 15 T 2 crosslinking PhastGel gradient 8 25 8 25 T 2 crosslinking PhastGel gradient 4 15 4 15 T 1 to 2 gradient crosslinker 32mm 0 112 M acetate 0 112 M Tris in both zones 6 4 43 x 50 x 0 45 mm 13 mm 32mm 0 112 M acetate and 0 112 Tris in both zones 6 4 75 T 3 C 20 T 2 C 6 0 t 3 C 12 5 T 2 C 85 Installation 86 PhastGel homogeneous 7 5 Stacking zone Separation zone PhastGel high density Stacking zone Separation zone PhastGel buffer strips Dimensions Volume Material uffer system ative strips DS strips H ative strips DS strips NDT WD WD Storage PhastGel sample applicators PhastGel sample applicator 12 0 3 sample wells well volume PhastGel sample applicator 8 0 5 sample wells well volume PhastGel sample applicator 8 1
42. The optimal temperature for too high this sensitive step is 30 3 The glutardialdehyde concentration 3 The optimal concentration is is too high 8 3 in solution 10 PhastSystem User Manual 80 1320 15 Edition AK Symptom Probable Cause Trouble Shooting Guide Solution s The gel turns yellow or brown upon drying Dark blotches around gel edges Very dark gel black or mirror effect Acetic acid wash ineffective in stopping the developer Touching the gel with fingers or metal objects Silver nitrate concentration is too high 1 Developer is too old 2 The gels were not developed long enough 3 The gel did not rotate in the solutions Check the concentration of acetic acid It should be 5 in water Increase the time for this step Avoid touching the gel surface with anything Use the tab of gel backing as a handle and use forceps to handle the gel Check the concentration use 0 5 or less silver nitrate in solution 1 Use fresh developer 2 Develop gels for 1 or 2 minutes longer 3 Check that the gel holder rotates during processing If it dose not call service Weakly stained bands PhastSystem User Manual 80 1320 15 Edition AK 11 Trouble Shooting Guide 12 PhastSystem User Manual 80 1320 15 Edition AK Ordering information and technical data 8 8 Ordering information and technical data 8 1 Ordering information 8 1 1 Gel media and acces
43. and bend the plastic tab up using the forceps This makes it easy to handle the gel Lower the gel onto one of the gel areas so that a film of liquid free from air bubbles forms between the gel support and separation bed Remove any air bubbles by sliding the gel around Finally position the gel so that its edges are in perfect alignment with the red lines Follow this procedure for the second gel 5 Remove any excess liquid with absorbent paper Note If only one gel is being run make sure that the empty gel area is dry 6 Use a pair of forceps to gently lift and peel the plastic film from the gel surface 7 Lower the electrode assembly Check that the inner anode nearest the cathode and the cathode have complete and even contact with the gel surface Run your thumb gently along the top of the electrodes 8 Lower the sample applicator arm and close the separation compartment lid Fig 20 Positioning a gel PhastSystem User Manual 80 1320 15 Edition AK 45 5 Operation 46 Cathode Inner anode Gel bed area Removing the plastic film Fig 21 Removing the plastic film Starting the run 1 Press SEP start stop and enter the number of gels for this run NUMBER OF GELS 0 lt do gt Methods are programmed for 1 gel If you enter 2 gels here the current and power will be adjusted automatically so that both gels run under the same conditions according to the programmed method only if both gels are
44. anium 42mm 37 mm inner electrodes IEF 43 mm outer electrodes native and SDS PAGE Electronically cooled heated by Peltier element 0 70 C Automatic with sample applicators If the separation compartment lid is open during a run an alarm will sound and the internal power supply is switched off 83 Installation 84 8 2 2 Development unit Dimensions Weight Capacity Agitation Development Number of ports Temperature control range time error Development chamber material volume Temperature sensor Level sensor Gel holder 8 2 3 Common data Safety regulations EMC Safety regulation Safety glass Operation environment room temp humidity 300 x 300 x 138 mm W x L x H 4 8 kg 1 or 2 gels Gels are rotated in solutions during development Solutions are automatically pumped into and out of the development chamber 9 ports are available for solutions to enter and exit the chamber through port 0 i reserved for waste Up to 50 C the chamber only heats lt 4 min to heat solutions from 20 to 45 C 4 C from programmed temperature once the programmed temperature is reached Stainless stee Approximately 70 ml of solution will be pumped into the chamber Stainless steel Enclosed in glass Stainless steel This product meets the requirements of the EMC Directive 89 336 EEC through the harmonized standards EN 50082 2 emission end EN 50082 1 immunity Note The
45. apacity The cooling capacity of the separation bed will depend on the following 1 the ambient temperature 2 the power applied to the gels and 3 if one or two gels are run Fig 4 below illustrates the separation bed temperature versus time for native PAGE SDS PAGE and IEF runs The running conditions are given in the caption under the graph A slight temperature drift can be seen for the IEF run with an ambient temperature of 28 C Even with an ambient temperature of 38 C no temperature drift is experienced with native gradient and SDS PAGE runs For 2 gels 20 18 Cooling bed temperature C 3 Native and SDS PAGE ambient temp 38 C 2 IEF ambient temp 28 C 1 IEF ambient temp 23 C 0 20 f 4o 60 80 100 Average run time Time min Fig 4 Separation bed temperature vs time The plots represent the following conditions I IEF PhastGel IEF 3 9 2000 V 5 mA 7 W 10 C with 23 C ambient temperature 2 Same as 1 but with an ambient temperature of 28 C and 3 Native gradient SDS PAGE PhastGel gradient media 400 V 15 mA 4W 15 C with ambient temperature of 38 C PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 Fig 5 below shows the lowest separation bed temperature maintained within I C for separations run at 4 and 7 watts with different ambient temperatures Lower temperatures can be achieved but temperature drifts exceeding I C might
46. ation is too high Add enough CuSO to make the solution 0 1 CuSo Do not recycle the fix and wash solutions more than 3 or 4 times The gels can be further destained even after drying Place the gels in the development chamber and start the destaining step 1 Recycle the fix and wash solutions no more than 3 or 4 times Destain the gel again 2 Check the concentration for the technique you are using 0 02 for IEF Trouble Shooting Guide PhastGel electrophoresis Symptom Probable Cause Solution s No or too little glycerol in the Rehydrate the gel in glycerol preserving solution acetic acid water according to Development Technique Files Dry it again Store the dried gel in a plastic Gels curls and or slide holder or cover the gel cracks after drying again with the protective film you removed prior to separation Protein 1 Too much glycerol in the last 1 Donor add more than bands preserving solution 10 glycerol fade after 2 The gel was exposed to direct 2 Store the gel in a fairly dark storage sunlight for an extended period place e g a notebook Silver staining 1 The developer solution is too old 1 Use fresh developer Check or it contains too much the concentration of the Dark yellow formaldehyde formaldehyde The optimal or brown concentration is 0 04 of background 37 aqeous formaldehyde i e really 0 015 formaldehyde in solution 2 The developer step temperature is 2
47. blue area in the center of the gel 1 Coomassie is not sensitive enough for the sample concentration applied to the gel 2 Not enough liquid in the development chamber during staining Weakly stained 3 Too low temperature for staining bands 4 The wrong method from the development method file was started by mistake 5 The gel did not rotate in the solutions 1 The stock solution is stable for 1 month Filter the stock solution before use Make up the final solution fresh for each day Do not recycle 2 The optimal staining temperature is 50 C Stain particles can be removed from the gel surface with a soaking wet cotton swab Gently rub the swab across the gel 3 Ensure out the tubing Do not allow coomassie solution to dry in the tubing 4 Rinse out the tubing Do not allow coomassie solution to dry in the tubing 1 The lower gel in the holder must face upwards 2 Filter the stock solution stable for one month 1 Try silver staining or concentrate the sample 2 Make sure the bottles contain at least 80 ml of solution and that the tubes are completely submerged in the solutions 3 Check the temperature sensor calibration see chapter on Maintenance in the System Guide 4 Check the method make sure it is the correct one and that it corresponds to the bottle and port number arrangement 5 Check that the gel holder rotates during processing If it does not call service
48. c film works well This film is sensitive to red thus blueand dark bands become more pronounced For UV illumination we recommend Polaroid 667 film with ASA 3000 or a similar film Filters for black and white Lens filters give increased band contrast and good color balance With UV illumination filters are necessary A list of filters that you may want to try when photographing gels is given below Common Type of Type of staining illumination filter technique fluorescent detection UV yellow or orange Coomassie or fluorescent deep yellow or red Coomassie like dyes try medium red with Panchromatic film green dyes fluorescent red silver fluorescent medium red PAS glycoprotein fluorescent blue or orange Paper A medium to hard paper with glossy finish will give best results for black and white photographs PhastSystem User Manual 80 1320 15 Edition AK Evaluation and presentation of data 6 6 2 Evaluation Procedures for measuring the isoelectric points and molecular weights of proteins using calibration proteins are described A brief discussion about evaluating PhastGel media with PhastImage is presented at the end Isoelectric point measurement Isoelectric points pl of proteins are conveniently and accurately measured using calibration proteins Calibration proteins indicate pH gradient profiles in gels By measuring the distance of a sample protein from a reference point to where it focuses its pl can b
49. ce 5 3 Running IEF media In IEF techniques a pre focusing step is usually run before the sample is applied This pre focusing time is programmed in the method as the volthours elapsed before the sample applicator is lowered onto the gel Start the method and load the sample applicator s during the prefocusing time Loaded sample applicators can be put into the applicator arm any time before the applicator arm goes down The general procedure for running PhastGel IEF media is described below See Separation technique file No 100 for running conditions and more specific information Preparing the gel compartment 1 Switch the system on and set the standby temperature to the temperature of the first step in the method you plan to run For details see Using the keyboard section 5 1 2 Lower the electrode assembly and sample applicator arm onto the separation bed Then press down both red eccentric levers until they click into place The electrode assembly is now in its lower position aligned evenly with the surface of the separation bed 3 Raise the electrode assembly to the vertical position 4 Fit the PhastGel IEF gel cover into place on the underside of the electrode assembly The electrodes will protrude through the slots Make sure the cover is aligned correctly by pressing firmly along the sides with your thumb Avoid touching the electrodes with your fingers skin proteins may distort results PhastSystem User Manual 80
50. clogged out port at the cursor position shown in step 2 above 5 Attach a syringe about 20 ml to the end of the clogged out port tube 6 Open the chamber lid and press do Pump air into and out of the out port tube with the syringe to dislodge the obstruction Note The channel between the port and the chamber is only open during this step If you do not succeed in unclogging the port during this EMPTYING step start the step again as described above To change the 10 port gasket If the chamber will not fill despite the above measures try changing the 10 port gasket Follow the directions on page 78 of the manual To continue the run press DEV pause continue The method will continue by FILLING the chamber 210 gt POWER FAILURE IN DEV UNIT e Check that the development unit is plugged into the wall outlet e Check that the development unit is properly connected to the separation and control unit via the communication cable e Check the fuses in the back of the development unit 211 gt POWER FAILURE METHOD S SET TO PAUSE If mains power fails longer than 5 10 seconds running methods will be paused When power returns an alarm will sound to inform you about the power failure You must press SEP pause continue or DEV pause continue for the method s to continue from where they left off When mains power fails less than 5 10 seconds runnings methods will automatically continue from where t
51. d and to activate the standby temperature PhastSystem User Manual 80 1320 15 Edition AK 29 3 Description of the system 30 Run control keys The key block on the far left of the keyboard is used to start stop pause continue and monitor separation and development runs These keys are described below G3 REAL CONDITION SEP DEV real real condition condition SEP stand by temp SEP DEV pause pause continue continue SEP DEV start start stop stop Fig 14 Run control keys SEP start stop Press to start a separation run Press again to end the run DEV start stop Press to start a development run Press again to end the run SEP pause continue Press to stop a separation run temporarily Press again to continue the run from where it left off DEV pause continue Press to stop a development run temporarily Press again to continue the run from where it left off When you pause a run the corresponding LED will blink At 20 second intervals a short alarm will sound to remind you that a method is paused Once you press a pause key the display will show the step number and the number of volthours or minutes that elapsed during that step for example SEP 1 2 PAUSE 68 Vh or DEV 1 08 PAUSE t 10 0 min During power failures running methods are automatically paused For power failures lasting less than 5 10 seconds methods will automatically continue when power is restored For longer p
52. d as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment PhastSystem User Manual 80 1320 15 Edition AK Help Message Reference Help Message Reference This section is designed as a reference for the help messages that appear on the display when you press the help return key or at an alarm condition Below help messages are listed by number they have on the display You will find explanations for some help message and references to further information concerning the help message In some instances suggestions are made to help you solve a particular problem for example if the development chamber cannot fill correctly IMPORTANT Once you have displayed a help message you must press help return again to return to previous display Key messages The following help messages will appear on the display when you press these keys and then press help return SEP start stop 1 gt METHOD ENDS AND VOLTAGE IS TURNED OFF If you press do the running method ends DEL 2 gt DELETE METHOD OR ONLY A METHOD STEP See page 26 of the manual insert 3 gt lt do gt ADDS A STEP BEFORE THIS STEP If you press lt do gt a step will be inserted before the step number appearing on the display See page 27 of the manual Copy 4 gt COPY A METHOD OR ONLY A METHOD STEP See page 2
53. declaration of conformity is valid for the instruments when it is e used in laboratory locations e used in same state as it was delivered from GE Healthcare AB except for alterations described in the user manual e used as stand alone unit or connected to other CE labelled GE Healthcare AB instruments or other products as recommend This product meets the requirements of the low Voltage Directive LVD 73 23 EEC through the harmonized standard EN 61010 1 Class 1 apparatus 4 40 C Max 95 PhastSystem User Manual 80 1320 15 Edition AK Electricity requirements mains voltage mains frequency power Power disturbance Mains failure Installation 100 120 V AC 120 model 220 230 240 V AC 220 V model 50 60 Hz Maximum 30 VA Separation and control unit 200 VA Development unit 330 VA together The system is protected against mains disturbance and static discharges For port failures lasting less than 5 10 seconds running methods will automatically continue when power i returned For power failures lasting more than 5 10 seconds running methods are set to pause an alarm sounds and a message appears on the display informing you about the power failure 8 2 4 PhastGel separation media and accessories A list of the technical data for PhastGel separation media and accessories is given below Common data Gel material Gel backing Storage PhastGel IEF media Dimensions Gel matrix Separation length
54. display and the keyboard In this section the keyboard will be described in key blocks the numerical pad the programming and editing keys and the run control and monitoring keys In this manual keys are always referenced by the key text in quotation marks Turning on the system The power on off button is placed at the back of the separation and control unit The microprocessor automatically runs a diagnostic test every time the unit is turned on The test includes the temperature sensors back up battery and level sensor When the system is turned on the display shows DIAGNOSTICS IN PROGRESS After a few seconds the display changes to DIAGNOSTICS SUCCESSFULLY COMPLETED or if an error is found an error message appears and an alarm sounds f no error is found the separation program mode i automatically selected The LEDs SEP ON and DEV ON are lit when a separation method or a development method is running If you pause a run the corresponding LED will blink as a remember The PROGRAM MODE LED is lit when you select one of the keys SEP method file of DEV method file for programming and when the system is turned on The REAL CONDITION LED is lit when a method is started or when you press one of the keys SEP real condition or DEV real condition One or more of the LEDs may be lit at one time since you can be running both a separation and development method while you are programming a method PhastSystem U
55. e Develop the protein bands using one of the techniques in the Development Technique File To easily measure the band distance mount the gel in a slide frame and project the image to the desired format using a slide projector Alternatively scan the gel with Phastlmage Measure the migration distance of the calibration proteins and calculate their R values distance of the band from the origin distance from the origin to the reference point Use the furthest migrating calibration protein as the reference point With SDS PAGE use the tracking dye position as the reference point PhastSystem User Manual 80 1320 15 Edition AK Evaluation and presentation of data 6 Plot the Rf values of calibration kit proteins against the logarithms of their molecular weight 7 Calculate the Rf value for the sample protein s Interpolate their corresponding log molecular weight from the calibration plot Fig 35 shows an example of calibration curves established using SDS denatured LMW calibration kit proteins PhastGel gradient 10 15 Fig 35 SDS denatured LMW low molecular weight calibration kit and chymotrypsinogen A run an PhastGel gradient 10 15 with PhastGel SDS buffer strips The gels were run according to the method in Separation technique file No 110 The kit proteins were reconstituted in 200 ul of SDS buffer PhastSystem User Manual 80 1320 15 Edition AK 6 73 6 Evaluation and presentation of data 74 MW 200
56. e process will be two times faster at 40 C than at 20 C Based on experimental studies of Coomassie staining with PhastGel media we have found that as a general rule the reaction rate will double for every 20 C rise in temperature When the Ct factors are plotted against temperature a Ct curve is obtained This curve is shown in Fig 30 Ct factor Ct Curves Reference point 0 10 20 30 40 50 Temperature C Fig 30 Example of a temperature compensation curve for development processes that double in rate for every 20 C rise in temperature The curve is made by plotting the temperature compensation factors Ct factors For 5 30 4 0 and 50 C 20 C is the reference temperature on which the curve is based PhastSystem User Manual 80 1320 15 Edition AK 61 5 Operation 62 How to use Ct factors Each development method in the method file has a Ct curve instruction where you program Ct factors for 5 30 40 and 50 C PhastSystem interpolates a Ct curve from these points and stores it as part of the method program For each development step you program the process time t as the time required for the step at 20 C You also program the temperature you want the step to be processed at the development chamber can heat solutions up to 50 C During a development run the actual temperature of the solution in the chamber is measured every second From the Ct curve PhastSystem obtains the Ct factor fo
57. e assembly may take up two horizontal positions depending on the setting of the two eccentric levers The lower PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 position is used for PhastGel IEF media where the inner electrodes the anode nearest the cathode and the cathode rest directly on the gel The higher position is used for PhastGel electrophoresis media where the outer electrodes rest on PhastGel buffer strips which are held in place on the gel by the PhastGel buffer strip holder The buffer strip holder like the IEF gel cover also serves to prevent gels from drying out during electrophoresis PhastGel buffer strips PhastGel separation media He Cee ee PhastGel sample applicators PhastGel sample well stamp PhastGel IEF gel cover PhastGel buffer strip holder Fig 3 Sample application Separation methods Nine separation methods are available for programming For each method you can program two sample application instructions for lowering and raising the sample applicators an extra alarm instruction and up to nine steps For each step the voltage current power separation bed temperature and duration of the step in volthours is programmed Before a separation method is started the sample applicator arm rests a few millimeters above the gels After a programmed interval during the run the applicator arm is lowered to apply the samples to
58. e interpolated from the pH gradient profile Three pl calibration kits are available from GE Healthcare Table 1 Each kit contains 8 11 proteins depending on the kit Table 1 Selecting the correct pl calibration kit for PhastGel IEF media pl pl range Number of PhastGel calibration covered markers for IEF kit by the kit PhastGel interval PhastGel EF4 6 5 Low pl 2 80 5 85 4 4 15 5 85 PhastGel IEF 5 8 High pl 5 85 10 25 4 5 85 7 35 PhastGel IEF 3 9 Broad pl 3 50 9 30 10 3 50 8 65 Procedure for pl measurement Instructions for pl measurement in PhastGel IEF media are given below 1 If you know the approximate pl of the protein of interest select a suitable PhastGel IEF gel If it is unknown use PhastGel IEF 3 9 2 Use the appropriate separation method for IEF described in Separation technique file No 100 chapter 9 3 Reconstitute one vial from the Low pl High pl or Broad pl calibration kit in 30 40 ul of distilled water for Coomassie staining and in 2 ml for silver staining Reconstituted kit proteins can be store frozen at 20 C 4 Start the run and apply the samples to the gel use the procedures presented in Separation procedures section 5 2 Apply calibration proteins between the sample lanes On one side of the calibration proteins apply the sample at the cathode and on the other side at the anode This will help to ensure that proteins are in equilibrium for pl measurements they should focus at the sa
59. e using temperature compensation enter the time required for this step at 20 C regardless of the temperature you program for this step See page 65 of the manual for more information DEV 1 01 IN 0 OUT 0 T 00 0MIN T 00 C 28 gt TEMPERATURE FOR THIS STEP You can enter values from 0 C to 50 C however the chamber can only heat liquids When starting development run START DEV METHOD 1 00 lt do gt 18 gt PROCESS WILL START AT THIS STEP If you do not enter a step number the run will start at step 1 PhastSystem User Manual 80 1320 15 Edition AK 3 Help Message Reference Programming error messages If an alarm sounds while you are programming or editing a method or if an alarm sounds when you start a method press help return to find out what the problem is One of the following messages will be displayed 101 gt ILLOGICAL SAMPLE APPLICATOR VALUES When starting a separation run with a method which has illogical sample applicator values for example if the sample applicator is programmed to go down after 22 Vh in step 1 and step 1 only has 20 Vh 102 gt METHOD 0 DOES NOT EXIST When you try to call up method 0 methods are numbered from 1 9 103 gt ILLOGICAL METHOD STEP PARAMETERS When trying to start a separation method with illogical parameters other than applicator movement for example O V 104 gt DEV PAUSE PROHIBITED IF DEV OFF When trying to pause a development run not started 105 gt SEP PAUSE PROHIB
60. eir corresponding pl s are lentil lectin basic 8 65 lentil lectin middle 8 45 lentil lectin acidic 8 15 horse myoglobin basic 7 35 horse myoglobin acidic 6 85 human carbonic anhydrase B 6 55 bovine carbonic anhydrase B 5 85 B lactoglobulin A 5 20 soybean trypsin inhibitor 4 55 amyloglucosidase 3 50 Molecular weight measurement Molecular weights of globular and SDS denatured proteins are easily measured using PhastGel homogeneous or gradient media and one of the GE Healthcare calibration kits Pour calibration kits are suitable for use with PhastGel media the high molecular weight HMW kit for native or SDS denatured proteins the low molecular weight LMW kit for SDS denatured proteins a high molecular weight calibration kit especially prepared for SDS runs with PhastSystem HMW SDS and a molecular weight kit intended for SDS runs of very small proteins or peptides PMW GE Healthcare calibration kit proteins come in convenient freeze dried mixtures The proteins are exactly characterized and highly purified Exact protein amounts per vial have been chosen to give bands of equal intensity on staining with Coomassie Table 2 below shows the molecular weight ranges covered by the calibration kits PhastSystem User Manual 80 1320 15 Edition AK 71 6 Evaluation and presentation of data 72 Table 2 Molecular weight calibration kits available for native and SDS electrophoresis ug marker Storage number Marker MW
61. electrodes after every run with a moist lint free cloth After running sticky samples such as blood you should remove the entire assembly for thorough cleaning under running water or as described under Dirty electrodes page 2 Se also page 42 in the System Guide Dilute the sample or reduce the volume applied ae aa See Aae eet w a v v wv wwe wv vsi Sa YV Smiling bands PhastSystem User Manual 80 1320 15 Edition AK Trouble Shooting Guide Symptom Probable Cause Solution s Proteins do not reach their pl Loss of enzymes activity Sample precipitates at the site of application 1 Sample focused for insufficient number of volthours 2 Gradient drift over focusing 1 Enzyme is inactive at its pl 2 Cofactor removed during focusing Sample is applied too close to its isoelectric point 1 Apply the sample at different sites Note the Vh s for coalescence Stop the run sooner Find the correct volthours for focusing as in 1 above Try changing the pH of the gel after the run in an appropriate buffer This might result in band diffusion however Add cofactors to the detection solution Try the other two application points PhastSystem User Manual 80 1320 15 Edition AK Trouble Shooting Guide PhastGel gradient and homogeneous media Symptom Probable Cause Solution s SDS buffer strips were used instead of
62. electrophoretic techniques native polyacrylamide gel electrophoresis PAGE in gradient or homogeneous gels SDS PAGE in gradient or homogeneous gels and isoelectric focusing IEF These gels can be combined for twodimensional techniques PhastGel media are made of polyacrylamide bonded to a transparent polyester backing The gel surface is covered with a plastic film which prevents drying and contamination This film must he peeled off directly before use that is after the gel has been positioned onto the separation bed and excess water has been removed PhastGel media are individually packaged in airtight envelopes Once removed from their package gels should be used immediately PhastGel chemicals include buffer strips for electrophoresis and a Coomassie type stain PhastGel Blue R PhastGel media and chemicals are described below See Separation procedures and Development procedures for detailed instructions for using these products PhastGel IEF media PhastGel IEF media are homogeneous 5 T 3 C polyacrylamide gels containing Pharmalyte carrier ampholytes Pharmalyte generates stable linear pH gradients with a smooth conductivity profile across the entire pH range which means that high field strengths of 500 volts cm and above can be used Three different PhastGel IEF media are available PhastGel IEF 3 9 4 6 5 and 5 8 PhastGel IEF media are run without buffer strips The histogram shown here illustrates the pH ranges o
63. elete only step 2 of method 1 DELETE SEP METHOD 1 2 lt do gt insert Press to insert a free step between two programmed steps Before you press this key you must be at the method step that follows the step you wish to insert For example if you want to insert a step between steps 3 and 4 in separation method 1 press SEP method file GET SEP METHOD 0 0 FREE 23456789 Enter 1 4 and do SEP 1 4 1500V 07 0mA 2 0W 10 C 0100Vh Press insert INSERT AT SEP METHOD STEP 1 4 lt do gt Press do to confirm SEP 1 4 0000V 00 0mA 0 0W 00 C 0000Vh Step 4 is now ready for programming When you insert a step your are actually moving all the steps after the inserted one down to create a free step for programming In the example above step 4 becomes step 5 step 6 becomes step 7 and so on The help return key help return press to display help messages after an alarm sound after you press a programming key or at every cursor position Important you must press help return again to return to the previous display The help messages that may appear on the screen are fully described in chapter 7 Trouble shooting The do key To prompt you to reconsider when making a few important commands you are required to press the do key for confirmation after the entry for example when starting or ending a method This key is also used to select characters when naming a metho
64. ethod method You can name a method before during or after you program a method Procedures for naming methods are given in chapters Separations procedures and Development procedures A short instruction is given here 1 Press SEP method file to name a separation method or DEV method file to name a development method 2 Press name method 3 Press step forward until the method number you want to name appears on the display PhastSystem User Manual 80 1320 15 Edition AK 27 3 Description of the system DEL 28 4 Press gt or 4 until the first character you want in the name appears in the parentheses on the right of the display Depress these keys for more than one second to move rapidly through the character selection 5 Press do to enter the character appearing in the parentheses Press CE to erase any character you entered by mistake 6 Repeat steps 4 and 5 to enter the rest of the characters in the name Editing keys As well as changing entries in a programmed method you may also copy or delete methods or steps in a method or insert steps into programmed methods This is done using the keys described below First you must enter the separation or development programming mode Press SEP method file or DEV method file copy press to copy a method or method step The following commands will appear on the display COPY SEP METHOD FROM 0 0 TO 0 0 lt dc gt
65. f PhastGel IEF media with respect to the pl distribution of 800 proteins See Technical data chapter 8 and Separation technique file No 100 chapter 9 for further details PhastGel electrophoresis media PhastGel electrophoresis media are used together with PhastGel buffer strips Buffer strips made of high quality agarose with low electroendosmosis and high purity reagents serve as buffer reservoirs to generate discontinuous buffer systems in the gels during a run During a separation proteins are first concentrated in a porous stacking gel zone they then move into the separation gel zone where they are separated according to size The migration distance of a protein is related to the logarithm of its molecular weight MW Molecular weights are easily estimated using one of the GE Healthcare molecular weight calibration kits See Evaluation and presentation of data chapter 6 for instructions Seven different gels for electrophoresis are available three for gradient gel electrophoresis and four for homogeneous gel electrophoresis The three gradient gels are PhastGel gradient 10 15 with a continuous gradient from 10 to 15 polyacrylamide PhastGel gradient 8 25 with a continuous gradient from 8 to 25 polyacrylamide and PhastGel gradient 4 15 with a continuous gradient from 5 15 total polyacrylamide and a 1 2 gradient cross linker Three of the homogeneous gels are PhastGel homogeneous 7 5 PhastGel homogeneous 12 5 and PhastGel hom
66. g If your gels curl after drying or storage soak them in 7 10 acetic acid with glycerol according to the Development technique files until they uncurl Mounting gels Dry gels can be mounted in slide frames in photo albums or in note books Slide frames must have a 37 x 37 mm inside perimeter which have a 50 x 50 mm outside perimeter to view all the bands in the gel These are medium format frames which can be purchased at your local photography shop Slide frames with glass or rigid plastic sheets to enclose the gel will prevent damage to the gel during storage PhastGel media are 43 x 50 mm and must be trimmed to 43 x 43 mm to fit into 50 x 50 mm slide frames Photographing gels Below are some general tips for obtaining good photographic results of PhastGel media Camera type Ordinary 35 mm or Polaroid cameras are well suited for photographing PhastGel media provided they have a close up lens or similar apparatus PhastSystem User Manual 80 1320 15 Edition AK 67 6 Evaluation and presentation of data 68 Light source We recommend using a light box with a daylight fluorescent and or a UV light source The top of the box should be opaque white plastic or glass For color photography light boxes should be color balanced to 5 800 K daylight with adjustable light intensity Light metering can be difficult with light boxes as the background Generally the aperture must be two to three f stops higher than the light meter
67. gain call for service WARNING For continued protection against fire hazard replace fuses only with the same type and rating of fuses se spare parts list section 8 2 Temperature sensor calibrating Like other sensing devices the temperature sensors should be checked every now and then and recalibrated if necessary The temperature sensors are calibrated as follows 1 Lift up the lids of the separation and development units and turn off the system the separation bed will be 1 to 2 C higher than ambient temperature when the system is on Allow the sensors to equilibrate to ambient temperature preferably overnight Make sure the ambient temperature remains relatively constant Alternatively use a thermocouple to measure the temperature of the separation bed and development chamber temperature sensor 2 Turn the system on and press SEP method file 3 Press 9 2 0 and 2 9202 ignore the wrong key alarm when you do this The display will show 9202 CALIBRATE SEP TEMP T XX C lt do gt 4 The temperature shown is the previous calibration temperature not the ambient temperature If you want to recalibrate the temperature sensor press CE to clear the old calibration value and then enter the current temperature of separation bed 5 Press do to confirm 9202 SEP TEMP SENSOR CALIBRATED PhastSystem User Manual 80 1320 15 Edition AK 75 7 Maintenance and trouble shoo
68. h native and SDS techniques The only operational difference is the use of PhastGel native or SDS buffer strips and sample preparation The general procedure for running PhastGel electrophoresis media is described below See Separation Technique Files No 110 111 112 120 121 and 130 for running conditions and more specific information for each technique Preparing the gel compartment 1 Switch the system on and set the standby temperature to the temperature of the first step in the method you plan to run first For details see Using the Keyboard section 5 1 2 Adjust the electrode assembly to the high position by pressing up on both red eccentric levers until they click into place 3 Raise the electrode assembly to the vertical position Remove the IEF gel cover 4 Wipe off the separation bed with a moist lint free cloth to remove dust or particles It is also advisable to wipe off the electrodes but this must be done gently and the cloth must not leave dust particles Avoid touching the electrodes with your fingers finger proteins may distort the results Note In order to obtain the best results possible we recommend frequent cleaning of the electrodes Even minor amounts of deposited impurities have been shown to sometimes affect the resolution and band pattern Positioning the gels 1 Place a drop of water or insulating fluid approximately 60 75 ul onto the middle of the gel area s outlined by the red lines on the separat
69. hastGel media can also be electrotransferred to an immobilizing membrane with the help of PhastTransfer This is a rapid economical and efficient method of blotting For more detailed information please contact your GE Healthcare representative Introduction Programming development methods is similar to programming separation methods The only differences are the parameters to be programmed You may program and save up to nine development methods Each method contains 20 steps available for programming For each step the following parameters are programmed e The IN port the port the solution will enter through Ports 1 to 9 can be used port 0 is reserved for waste e The OUT port the port the solution will exit through Ports O to 9 can be used e The duration of the step t in minutes Each step can be up to 99 9 minutes e The actual temperature T the step will be processed at the maximum temperature is 50 C The development chamber can only heat solutions 52 PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Each method also contains a special programming option called temperature compensation that works in conjunction with temperature control This function does not operate unless you program it Temperature control When a solution enters the development chamber it is heated to the programmed temperature for that step The time it takes to heat the solution depends on the solution s initial tempera
70. he Ct curve otherwise go directly to step 11 below Programming an alarm 11 Press step forward for the alarm instruction 12 As for separation methods you can program an alarm to sound at a certain time during the method first enter the step number during which the alarm will sound for example step 10 EXTRA ALARM TO SOUND AT 7 10 t 00 0min 13 Press gt and enter the time when the alarm will sound during the step for exampie after 7 0 minutes in step 10 EXTRA ALARM TO SOUND AT 7 10 t 07 0min Programming method steps 14 Press step forward and enter the port number that the first solution will enter through enter 1 to 9 for example enter port 1 DEV 7 01 IN 1 OUT 0 t 00 0min T 00 C 15 Press and enter the port number the first solution will exit through enter O to 9 for example enter port 1 to recycle solution 1 DEV 7 0 IN 1 OUT 1 t 00 0min T 00 C PhastSystem User Manual 80 1320 15 Edition AK Operation 5 16 Press gt and enter the process time for this step for example enter 10 5 minutes nn you must press although it is shown DEV7 01 IN 1 OUT 1 t 10 5min T 00 C Note Ifyou are using temperature compensation you must program the process time as the time required for this step at 20 C regardless of the temperature you program for the step 17 Press and enter the actual temperature you want the step processed at fo
71. he chamber If the same problem occurs for this filling step call for service 209 gt CLEAN LEVEL SENSOR IN DEV CHAMBER This message will appear before a FILLING step When this message appears first open the chamber lid to see if the chamber is full or empty If the chamber is empty this message means the level sensor needs cleaning or it is damaged If the chamber is full this means that the out port in the previous step is clogged the liquid from previous step could not be pumped out or the 10 port valve gasket has swelled at this put port position Follow the directions below to correct this problem To clean the level sensor Gently wipe off the level sensor enclosed in glass on the underside of the development chamber lid with a moist cloth The level sensor is fragile and should be handle with caution PhastSystem User Manual 80 1320 15 Edition AK Help Message Reference To unclog ports 1 Press DEV real condition to see what step the run is at Then find out what the out port number was for previous step 2 Press SEP method file and the keys 9 5 0 and 1 ignore the wrong key alarm 9501 EMPTY TO PORT 0 lt do gt 3 First you must empty the chamber through a port that is clogged Enter the number of a suitable port to empty the chamber through Close the chamber lid and press do when you are ready 4 When the chamber has emptied press CE and enter the number of the
72. he lid in the development chamber enclosed in stainless steel These are calibrated before shipment but you may want to check them before using PhastSystem The sensors can be checked and calibrated individually See the chapter on Maintenance for instructions 4 4 Before use Before using the development unit we recommend that you run a cleaning method to remove dust accumulated during storage and shipment A cleaning method requires only distilled water and the level sensor shield instead of gels The level sensor shield is in the gel holder in the development chamber when you receive PhastSystem See chapter 5 the section on cleaning method for instructions The level sensor shield must remain in the chamber when running methods without gels otherwise the chamber will not fill WARNING The level sensor on the underside of the development chamber lid is enclosed in glass and is quite fragile Use extreme care when cleaning this sensor PhastSystem User Manual 80 1320 15 Edition AK 35 4 Installation 36 PhastSystem User Manual 80 1320 15 Edition AK Operation 5 5 Operation The aim of this chapter is to show you how to program a separation method and a development method how to load samples into the sample applicators and how to run PhastGel IEF media electrophoresis titration curves and PhastGel homogeneous and gradient media 5 1 Programming separation procedures Introduction Nine separation me
73. he time intervals listed above will depend on the technique that is run PhastSystem User Manual 80 1320 15 Edition AK 7 1 Introduction This users guide includes the following chapters Chapter 2 Important safety information Chapter 3 Description of the system introduces you to PhastSystem Chapter 4 Installation tells you how to install PhastSystem Chapter 5 Using the keyboard prepares you for programming and running methods Separation procedures shows you how to program and run separation methods Development procedures shows you how to program and run development methods Chapter 6 Evaluation and presentation of data gives advice on drying mounting and photographing gels and describes procedures for molecular weight and isoelectric point measurement using calibration proteins Chapter 7 Maintenance and trouble shooting shows you how to replace and clean certain parts of the instruments and how to calibrate the temperature sensors Provides current trouble shooting recommendations If you have any problems during programming or operation you find all help messages listed here Chapter 8 Ordering information and technical data gives you all information needed to order the products mentioned in this manual You will also find a list of the most common spare parts required for maintenance of PhastSystem A list of the technical data on PhastSystem instruments and PhastGel media and accessories is also inc
74. hey were stopped once power is returned 212 gt TEMP SENSOR NOT CALIBRATED Calibrate the temperature sensors according to the instructions given on page 77 of the manual PhastSystem User Manual 80 1320 15 Edition AK 7 Help Message Reference System error messages The following messages will appear on the display if an error is detected during diagnostics when you turn the system on 301 gt BATTERY NOT CONNECTED 302 gt BATTERY VOLTAGE TOO LOW 303 gt SEPARATION TEMP SENSOR NOT OK 304 gt DEVELOPMENT TEMP SENSOR NOT OK 305 gt PROM CHECKSUM NOT OK 306 gt PROCESSOR INTERNAL RAM NOT OK 307 gt EXTERNAL RAM NOT OK 308 gt PROCESSOR TIMERS NOT OK 309 gt AD CONVERTER NOT OK First try turning the system on and off several times Fluctuations in the main power may cause a false error to appear on the display If the error message still appears note down the message and call for service PhastSystem User Manual 80 1320 15 Edition AK Trouble Shooting Guide Trouble Shooting Guide This guide lists the symptoms in illustrated form when possible of some problems you might encounter with separation and development techniques Probable causes are given along with solutions to correct or prevent the problems The symptoms drawn in gel format are amplified so you can easily identify them and refer to them Some symptoms such as missing bands are impossible to illustrate so the symptoms is described in the gel outline This guide
75. hode for each gel The electrodes are made of platinized titanium An assembly with reversed polarity is also available for electrophoresis of basic proteins in their native state A high voltage power supply inside the separation and control unit generates the required electric field for electrophoresis See power supply page 13 If the lid is opened during a run the high voltage supply switches off automatically to eliminate electrical hazard An alarm will sound until the lid is closed or until the run is paused em p Separation compartment Fig 2 Separation compartment Sample application Samples are applied to gels with PhastGel sample applicators These small comb like pieces have a series of capillary wells Samples are drawn into the capillaries and held there until the applicator is lowered onto the gel at a set time in the program Once the applicators are loaded with samples they are placed into one of the slots in the sample applicator arm The sample applicator arm has four alternative sample applicator positions for each gel The position nearest the cathode is for PhastGel electrophoresis media and the other three positions are for PhastGel IEF media The plunger toward the back of the compartment holds the applicator arm up until it is time for sample application The electrode assembly and the applicator arm are raised so the gels can be positioned onto the separation bed When lowered again the electrod
76. ia Tel 60 3 8024 2080 Fax 60 3 8024 2090 e Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 e Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 e USA Tel 800 526 3593 Fax 877 295 8102 imagination at work User Manual 80 1320 15 AK 03 2006 E Elanders sterv la 2006
77. ical hazard An alarm will sound until the lid is closed or until the run is paused PhastSystem User Manual 80 1320 15 Edition AK 11 2 Important safety information 2 3 Safety precautions The voltage supplied by PhastSystem is capable of delivering a lethal electric shock The numerous safety devices and circuits built into the instrument prevent this The pause and start stop keys can also be pressed to halt the supply of power at any stage of the experiment or operation of PhastSystem Nevertheless in Keeping with good laboratory practise we advice you to take the following precautions when dealing with the instrument 1 Regularly check all insulation cables take care not to damage the units especially the separation compartments lid Note For full safety it is important that the lid is not tampered with 2 Ensure that the mains cables are plugged into fully grounded mains outlets 3 Allow only authorized service representatives to service or work on the electrical circuitry of PhastSystem 4 Avoid spilling buffers or other conduction liquids onto the instrument 5 Allow the ventilation slots situated at the rear of PhastSystem to have free access to a good flow of air 12 PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 3 Description of the system The aim of this chapter is to introduce you to PhastSystem Each component of PhastSystem is described in turn the separation
78. indicates depending on the meter type Thick black paper can be placed around the gel on the light box to eliminate excess back lighting Positioning gels on the light box Wet gels photograph better than dry ones Dry gels can be soaked in 7 10 acetic acid until they rehydrate Small particles and fibers can be removed from the gel surface with a soaked cotton swab Gently run the swab across the gel For light boxes with a fluorescent light source position the gels as follows Place the gel in a transparent glass petri dish and cover the gel with 1 to 2 mm of 7 10 acetic acid Remove any air bubbles by sliding the gel around Set the dish on the light box Alternatively position the gels as you would position them on the separation bed For light boxes with a UV light source the gels must be positioned gel side down onto the light box because the gel backing absorbs the UV light The emitted light from the protein bands will pass through the gel backing Film For fluorescent illumination we recommend positive negative Polaroid 665 film with ASA 75 or a similar film The negative from this film will produce better prints than the positive So when your are satisfied with the results from the positive have the negative developed for the final print for example if you plan to publish your results The shutter speed is slow for this film so your camera should be mounted on a tripod or stand For 35 mm cameras a fine grain Panchromati
79. ion bed 2 Take one or two gels from the refrigerator Use a pair of scissors to cut the package along three sides Remove the gel from its package with a pair of forceps use the plastic tab of the gel backing as a handle The thin plastic film on the gel surface protects the gel from contaminants and from drying and should be left on for now 3 Use a waterproof pen to mark the underside of the gel for identification You might have to wipe the back of the gel first 4 Place the gel on a hard surface and bend the plastic tab up using the forceps This makes it easy to position and remove the gel from the bed Lower the gel onto one of the gel areas so that a film of liquid free from air bubbles forms between the gel support and separation bed Remove any air bubbles by sliding the gel around PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Fig 23 Bending the tab up Finally position the gel so that its edges are in perfect alignment with the red lines Follow this procedure for the second gel Ca Gel bed area o Fig 24 Positioning a gel 5 Remove any excess liquid with absorbent paper Note If only one gel is being run make sure that the empty gel area is dry 6 Use a pair of forceps to gently lift and peel the plastic film from the gel surface Eccentric levers in upper position Removing the plastic film Fig 25 Removing the plastic film PhastSystem User Manual 80 1320 15 Editio
80. it PhastSystem User Manual 80 1320 15 Edition AK Help Message Reference 117 gt CANNOT DELETE A RUN RUNNING STEP When trying to delete a step that has run or a step that was running when the method was paused Steps can be deleted in a running method only if they follow the paused running step 118 gt TO INSERT ANEW STEP DO lt pause gt When trying to insert a step in a running separation or development method without first pressing SEP pause continue or DEV pause continue 119 gt CANNOT INSERT AT A RUN RUNNING STEP When trying to insert a step before or at the step in progress when you paused the run Steps can be inserted into a running method only if they follow the paused running step 120 gt ILLOGICAL TEMP COMPENSATION CURVE When starting a development run with a method containing a temperature compensation Ct factor 0 0 or a Ct curve that decreases with increasing temperature The temperature compensation curve instruction i a programming option If you do not want temperature compensation make sure all the Ct factors are set 1 0 for 5 C 30 C 40 C and 50 C See page 55 of the manual for more information 121 gt MAx STANDBY TEMP IS 70 C When trying to enter a standby temperature greater than 70 C 122 gt MAx DEV TEMP IS 50 When trying to enter a development temperature greater than 50 C 123 gt SEPARATION LID IS NOT CLOSED When trying ti start or continue a separation run with the separa
81. ke sure the sample does not contain air bubbles PhastSystem User Manual 80 1320 15 Edition AK PhastGel IEF media Symptom Probable Cause Trouble Shooting Guide Solution s 1 Particulates in the sample N Poorly soluble protein that precipitates when applied to the gel i 3 The sample has dried and Streaking aggregated in the applicators before being applied to the gel The aggregates are left on the gel and leak protein during the run 4 Dirty sample applicators 5 Sample overloading 1 The fixing solution was too old 2 The proteins in the gel were not fixed soon enough after the separation Diffuse bands 1 Uneven electrode contact with the gels 2 Dirty electrodes Uneven iso pH lines The buffer capacity of the sample is too high 1 Centrifuge or filter samples 2 Try applying the sample at a different point Decrease the field strength for sample application e g 200 V and 2 5 mA or even less 3 Load the samples just prior to the sample application step especially blood and serum samples 4 We recommend that sample applicators be used only once 5 Dilute the sample 1 Recycle the fixing solution no more than 3 to 4 times 2 Bands will start to diffuse immediately after the method is stopped fix the proteins in the gel as soon as possible 1 Before starting a run press along the electrodes to ensure good contact with the gel 2 Clean the
82. l condition the number of elapsed volthours Vh for the step which is running is displayed Press SEP real condition to display A Vh again PhastSystem User Manual 80 1320 15 Edition AK 51 5 Operation During the course of the run you may at any time start a development run for a finished gel or program another separation or development method To go back and check on the separation run in progress press SEP real condition Stopping the run 1 When the alarm sounds check to see that the tracking dye has reached its proper distance from the electrode for SDS PAGE If it hasn t the method can be allowed to proceed until it does The alarm is temporarily stopped by pressing SEP real condition 2 To stop the method press SEP start stop PRESS lt do gt TO END SEP METHOD 3 Press lt do gt to confirm The display will show the temperature of the separation bed and the accumulated volthours for the method just ended SEP OV 0 0mA 0 0W 18 C 300 AVh Proceed with development immediately after the method is stopped 5 5 Programming development procedures This section describes the procedures for programming and running development methods First an introduction to programming is given including a section on temperature control Then a step by step instruction for programming development methods is given followed by the procedure for running the methods Finally temperature compensation is described P
83. l is close to the gel s edge positioned correctly within the red vertical outline on the bed Occasional Sample list on the plastic when Take care you when you empty lanes you put the sample applicator insert the sample applicator in the slot especially when using the buffer strip holder PhastSystem User Manual 80 1320 15 Edition AK 1 Trouble Shooting Guide Symptom Probable Cause Solution s egida SALVIE Ce etyeus ETS ENT H Wavy bands EATE 2 Ba ee oF ENVA CCHIT Local band disturbance MeL Eeyore CE REE EEEL r Wavy bands at or near the point of sample application Dotted or slashed bands Dirty electrodes Air bubbles present between the gel and the bed Too much salt in the sample 1 Field strength too high for sample application 2 Air bubbles in the sample Clean the electrodes with a wet lintfree cloth after every run From time to time pull out the electrode assembly and wash the electrode with detergent or HNO using a soft brush Rinse thoroughly with distilled water Airdry or dry with a hair dryer Use about 60 75 ul of water to position the gels on the bed Remove all air bubbles between the gel and the bed Dilute or desalt the sample For IEF try another sample application position 1 Decrease the field strength for the sample application step use 220 V 2 5 mA for IEF and 400 V 1 0 mA for native PAGE 2 Ma
84. lder CS J P Development chamber Fig 6 Development chamber PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 PVC tubing Cap set Tube markers 10 Port valve Fig 7 10 port valve Chemical resistance The parts that come into contact with development solutions in the development unit are resistant to chemicals typically used in Coomassie and silver staining for example acetic acid methanol and silver staining solutions If you plan to use other chemicals for example to clean the unit you should first check the resistance of the wetted parts to the chemical in question The chemical resistance of a polymer depends on many factors including the temperature and concentration of the solution the application a compound that swells may function well as a static seal yet fail in dynamic applications and the period of exposure Table 1 below is intended as a general guide for the chemical resistance of the wetted parts in the development unit If you are in doubt about the resistance of wetted parts to a certain chemical test the parts first order spare parts for such tests see Ordering information chapter 8 In general you should avoid using ketones hot strong acids and organic hydrocarbons PhastSystem User Manual 80 1320 15 Edition AK 19 3 Description of the system 20 Table 1 A general guide for the chemical resistance of the wetted parts in the deve
85. lopment unit Wetted parts Material of Generally Generally construction resistant to attacked by Distributor and PVDF strong acids and ketones esters and hot distributing plate PVDF bases in moderate acids concentration and alcohols and hydrocarbons Gasket 10 port fluoro rubber moderate acids hot strong acids esters valve strong bases ketones and bleach many solvents alcohols aldehydes Tubin 10 port to chamber Tubing bottles to 10 port valve Chamber gel holder and temp sensor Chamber lid gasket Chamber lid Teflon PVC stainless steel EPDM pps most chemicals strong acids and bases in moderate concentration alcohols aldehydes and bleach most chemicals strong acids and bases alcohols aldehydes and ketones strong acids in moderate con centration in high concentration alcohols aldehydes and ketones extreme conditions hot acids ketones and hydrocarbons long exposure to salt solutions hot acids and aromatic hydrocarbons hot strong acids aromatic hydrocarbons and bleach 1 These parts are illustrated on pages 16 17 and 79 2 Polyvinylidine fluoride 3 Polyvinyl chloride 4 Ethylene propylene copolymer and terpolymer 5 Polypropylene or polypropene PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 3 3 PhastGel media and chemicals At present PhastGel media are available for four types of
86. lopment unit 60 PhastSystem User Manual 80 1320 15 Edition AK Operation 5 5 8 Temperature compensation Some development techniques may contain steps that are extremely short and or sensitive to temperature variations of the incoming solutions PhastSystem has a temperature compensation function that can be programmed to adjust for these variations This function is based on the rate of development processes at 20 C PhastSystem uses 20 C as the reference temperature During a development run deviations from 20 C are compensated for by adjusting the programmed process time t If the temperature of the solution in the development chamber is above 20 C the process time will be reduced Conversely if the temperature is below 20 C the process time will be extended The degree of compensation is determined by the temperature compensation curve Ct curve programmed for the method The Ct curve is programmed with four values called temperature compensation factors Ct factors What Ct factors are Ct factors describe the rate of a process at a certain temperature relative to the rate of that process at 20 C Thus the Ct factor for any process run at 20 C is 1 0 Ct factors are greater than 1 0 for temperatures above 20 C and are less than 1 0 for temperatures below 20 C For example if a method takes 30 minutes at 20 C and 15 minutes at 40 C the Ct factor for that method at 40 C is 2 0 that is the rate of th
87. luded Chapter 9 Separation technique files you will find optimized methods for a number of separation techniques Development technique files you will find optimized methods for a number of development techniques Application notes here you can file application and technical notes covering specific techniques or application areas A technological extension of PhastSystem is PhastTransfer PhastTransfer PhastTransfer brings speed reproducibility and convenience to semidry electrophoretic transfer of proteins from PhastGel separation media to immobilizing membranes The small format of the gels together with semi dry transfer method minimize the amount of reagents needed for detection Elution efficiency is greater than 90 for most protein systems At 1 0 mA cm high transfer recovery is obtained usually within 10 30 minutes PhastSystem User Manual 80 1320 15 Edition AK Introduction 1 Phastsystem Fig 1 PhastSystem consists of a separation and control unit a development unit PhastGel separation media accessories and a technical support package PhastSystem User Manual 80 1320 15 Edition AK 9 1 Introduction 10 PhastSystem User Manual 80 1320 15 Edition AK Important safety information 2 2 Important safety information WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and
88. me point in the gradient PhastSystem User Manual 80 1320 15 Edition AK 69 6 Evaluation and presentation of data 70 5 After electrophoresis develop the protein bands according to one of the development methods given in chapter 9 Development Technique Files 6 To easily measure the band distance mount the gel in a slide frame and project the image to the desired format using a slide projector Alternatively scan the gel 7 Plot the known pl value of each pl calibration protein versus its distance from a reference point e g the cathode to the nearest 0 05 cm Draw a line through the points to obtain the pH gradient profile of the gel 8 Measure the distance from the reference to the proteins of interest Use the pH profile of the gel to interpolate the pl points of these proteins Figure 1 shows an example of a pH gradient profile established using the Broad pl calibration kit Fig 33 Broad pl calibration kit run on PhastGel IEF 3 9 The gel was run according to the method in Separation Technique File No 100 The kit proteins were reconstituted in 35 ul of distilled H O PhastSystem User Manual 80 1320 15 Edition AK Evaluation and presentation of data 6 pH 0 10 20 30 40 Distance from cathode mm Fig 34 The pH gradient profile indicated by the Broad pl calibration kit for the gel shown in Fig 33 The gel was projected onto a 25 x 25 cm format for band measurement The proteins starting from the cathode and th
89. n AK 49 5 Operation 50 Place the PhastGel buffer strip holder over the gels by first sliding the buffer strip holder forward so that the two black pins and the holes in the holder form a hinge Lower the buffer strip holder onto the separation bed we Outer anode Pins Positioning the buffer strip holder Fig 26 Positioning the buffer strip holder 10 11 12 Take a pack of native or SDS buffer strips from the refrigerator Peel back the foil over two buffer strips and remove them with a spatula Note Gloves should be worn when handling buffer strips to prevent eventual disturbance from finger proteins Insert the buffer strips into the compartments in the buffer strip holder one in the anode and one in the cathode compartment Repeat this for the second gel Gently press down on them to ensure good contact between the buffer strips and the gel Buffer strips will protrude above the compartments by about 1 2 mm Lower the electrode assembly so that the outer electrodes the cathode and the anode furthest from it rest evenly on the buffer strips Gently press down along the top of the electrodes The electrodes must have complete and even contact with the buffer strips Lower the sample applicator arm Sample application L 2 3 Load the sample applicator s as described on page 39 Slide the loaded sample applicator s into the slot nearest the cathode Close the separation compartment lid
90. od A step by step instruction for programming a separation method is given below Remember that help messages can be accessed at any cursor position by pressing help return Selecting a method 1 Press SEP method file The method numbers that are free for programming are displayed in the parentheses GET SEP METHOD 0 0 FREE 123456789 2 Enter the number of a free method before the period method 3 will be used in this and the following examples GET SEP METHOD 3 0 If method 3 had a name the name would now appear in the parentheses Press do 3 The display will show METHOD 3 NAME The name field will he blank because you have not named the method yet You can name the method whenever you choose Naming a method 4 Press name method The display will show SEP 1 L A 5 Press step forward twice to display the name field for method 3 SEP 3 L A PhastSystem User Manual 80 1320 15 Edition AK Operation 5 6 Press gt or lt 4 until the first character you want in the name appears in the parentheses to the right for example method 3 can be called IEF 3 9 SEP 3 _ I 7 Press do to enter this character into the name field SEP 3 Le I 8 Continue with steps 5 and 6 above to enter the rest of the characters A method name can have up to 10 characters SEP 3 IEF 3 9_ 9 To continue programming to method 9 Press SEP method file the method number
91. of a free method Method 7 will be used in this and the following examples GET DEV METHOD 7 00 If method 7 had a name the name would now appear in the parentheses The positions after the period are for entering a step number when you want to go directly to a particular step 3 Press do to confirm The display will show METHOD 7 NAME The name field will be blank because you have not named the method yet You can name a method whenever you choose PhastSystem User Manual 80 1320 15 Edition AK 53 5 Operation 54 Naming a method 4 Press name method DEV 1 L A 5 Press step forward six times to display the name field for method 7 DEV 7 i A 6 Press gt or until the first character you want in the name appears in the parentheses to the right for example method 7 can be called COOM IEF Coomassie for IEF runs DEV 7 i C 7 Press do to enter this character DEV7 C_ C 8 Continue with steps 6 and 7 until all characters are entered DEV 7 COOM IEF_ F To continue programming the method 9 Press DEV method file 7 and do to select method 7 again DEV 7 NAME COOM IEF The name of the method will now appear on the display Programming the Ct curve 10 Press step forward for the Ct curve instruction DEV 7 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 If you plan to use temperature compensation follow the instructions on page 61 to program t
92. ogeneous 20 with a concentration of 7 5 12 5 and 20 polyacrylamide respectively The fourth homogeneous gel is PhastGel high density which has a polyacrylamide concentration of 20 and a 30 concentration of ethylene glycol See Technical data chapter 8 and Separation technique files 111 112 120 121 and 130 chapter 9 for further discussion and details PhastSystem User Manual 80 1320 15 Edition AK 21 3 Description of the system 22 12 Relative abundance PhastGel IEF 4 6 5 PhastGel IE PhastGel IEF 3 9 3 4 5 6 7 8 9 10 11 Fig 8 The approximate pH ranges of PhastGel IEF media are superimposed on a histogram showing the isoelectric point The histogram is made up of data from 800 proteins Gianazza E Righetti P G J Chromatography 193 1980 1 8 By kind permission of the authors and publisher The two histograms shown here illustrate the molecular weight ranges for PhastGel gradient media with respect to the molecular weight distribution of proteins in both denatured and non denatured form Denatured proteins 15 Relative abundance PhastGel Gradient 10 15 PhastGel Gradient 8 25 Subunit molecular weight x 10 Fig 9 The approximate molecular weight separation ranges of PhastGel gradient media are superimposed on a histogram showing the molecular weight distribution of denatured proteins The histogram is made up to data collected from 530 proteins Each ba
93. ose or damaged Replace the gasket if it is damaged See page 80 of the manual e If you suspect an obstruction in the tube between the 10 port valve and the development chamber follow the instructions given for help message 209 below for unclogging ports Note These instructions are for unclogging the put port but the same instructions are valid for unclogging the in port e Dismantle and clean the 10 port valve according to the instructions given on page 80 of the manual If this valve leaks change the gasket To continue the run press DEV pause continue The method will continue by FILLING the chamber Full chamber If the chamber is full of liquid when this message appears on the display it means that the lever sensor is not functioning properly First you must empty the chamber and the expansion chamber inside the unit it protects the pneumatic pump which is probably full of liquid Use the following instructions 1 Press SEP method file 2 Press keys 9 5 0 and 1 ignore the wrong key alarm 9501 EMPTY TO PORT 0 lt do gt 3 Close the chamber lid and press do The expansion and development chambers will empty through port 0 Or enter another port number at the cursor position 4 Check the level sensor If its broke call for service 5 Clean the level sensor with a moist cloth Be careful not to damage it 6 Press DEV pause continue The run will start by FILLING t
94. ounds automatically at the end of the method PhastSystem User Manual 80 1320 15 Edition AK 39 5 Operation 40 Programming method steps 15 Press step forward to program the first method step SEP 3 1 0000V 00 0mA 0 0W 00 C 0000Vh 16 Enter the limiting voltage up to 2000V for the first step in the method SEP 3 1 2000V 00 0mA 0 0W00 C O000Vh 17 Press gt to move the cursor to the next field and enter the limiting current up to 50 0 mA for the first step SEP 3 1 2000V 02 5mA 0 0W 00 C 0000Vh 18 Press and enter the limiting power up to 7 0 W for the first step SEP 3 1 2000V 02 5mA 3 5W 00 C 0000Vh 19 Press and enter the temperature of the separation bed for the first step see page 14 cooling capacity SEP 3 1 2000V 02 5mA 3 5W 15 C 0000Vh 20 Press gt and enter the duration of the first step in volthours up to 9999 Vh per step SEP 3 1 2000V 02 5mA 3 5W 15 C 0500Vh Note You can press 4 to go back to a field to change an entry 21 Press step forward to program the second step SEP 3 2 OOOOV 00 0mA 0 0W 00 C 0000Vh The second and the following steps in the method are programmed in the same way as step 1 In this example step 2 is left blank that is method 3 contains only one step After 9 steps in the method the display will show END OF METHOD In summary for the above example the sample applicators will be lowered onto the gels after
95. ower failures methods will remain in pause until you press SEP pause continue or DEV pause continue The display will show PhastSystem User Manual 80 1320 15 Edition AK SEP real condition DEV real condition Description of the system 3 POWER FAILURE METHOD SET TO PAUSE An alarm will sound and the LED will blink to inform you about the power failure SEP standby temp Press to cool or heat the separation bed before starting a separation A separation method does not start until the separation bed temperature for the first step is reached The standby temperature enables you to have the separation bed at a given temperature also when not running a separation This way you gain some time at the start of a separation The display will show SEP T 22 C Tstandby 00 C OFF lt do gt T is the actual temperature of the bed At the cursor you enter a standby temperature between 0 and 70 C most separations take place at 15 C Press do to turn the standby temperature on or off The monitoring keys SEP real condition Press to monitor the progress of a running separation method DEV real condition Press to monitor the progress of a running development method Press SEP real condition to monitor the progress of the entire method for example if method 1 is running the display may look like this SEP 1 3 1500V 02 0mA 3 0W 12 C 0500AVh On the display AVh is the n
96. p return to display information about a field marked out with the cursor When programming a development method GET DEV METHOD 0 00 FREE 123456789 19 gt MAX NUMBER OF DEV METHODS IS 9 GET DEV METHOD 0 00 FREE 123456789 20 gt MAX NUMBER OF DEV METHODS IS 20 DEV 1 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 21 gt TEMP COMPENSATION FACTOR AT 5 C DEV 1 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 22 gt TEMP COMPENSATION FACTOR AT 30 C DEV 1 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 23 gt TEMP COMPENSATION FACTOR AT 40 C DEV 1 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 24 gt TEMP COMPENSATION FACTOR AT 50 C For more information on temperature compensation see page 63 of the manual EXTRA ALARM TO SOUND AT 2 00 t 00 0min 14 gt PROGRAM AN EXTRA ALARM As in separation methods you can program an alarm to sound any time during the method First enter the step number and then the time t when the alarm will sound during the step When programming development steps DEV 1 01 N 0 OUT 0 T 00 0MIN T 00 C 25 gt THE LIQUID ENTERS THROUGH THIS PORT Choose a port from 1 to 9 port 0 is reservedfor waste DEV 1 01 IN 0 OUT 0 T 00 0MIN T 00 C 26 gt THE LIQUID EXITS THROUGH THIS PORT Choose a port from 0 to 9 DEV 1 01 IN 0 OUT 0 T 00 0MIN T 00 C 27 gt t IS THE PROCESSING TIME Enter the rime required for this process step at the temperature you program for this step If you ar
97. pany hcare Bio Sciences AB tan 30 SE 751 84 Uppsala Sweden hcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Hea Amersh GE Hea tl a tl hcare UK Ltd m Place Little Chalfont Buckinghamshire HP7 9NA UK hcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Hea tl hcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacfic Tel 852 2811 8693 Fax 852 2811 5251 e Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 e Austria Tel 01 57606 1619 Fax 01 57606 1627 Belgium Tel 0800 73 888 Fax 03 272 1637 e Canada Tel 800 463 5800 Fax 800 567 1008 Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1 985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 e France Tel 01 69 35 67 00 Fax 01 69 41 96 77 Germany Tel 0761 4903 490 Fax 0761 4903 405 Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 Netherlands Tel 0165 580 410 Fax 0165 580 401 Norway Tel 815 65 555 Fax 815 65 666 Portugal Tel 21 417 7035 Fax 21 417 3184 e Russia amp other C S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 e South East As
98. r example 50 C DEV 7 01 IN I OUT 1 t 10 5min T 50 C The maximum temperature you can program is 50 C The chamber can only heat solutions but you can program values lower than the incoming solution s temperature if you do not want the solution heated Press step forward to program any subsequent steps After step 20 the display will show END OF METHOD Press step backward to go back through the method to double check the parameters Editing a method To edit a programmed method press DEV method file and select the method you wish to edit You can select the step you want to change by entering the step number after the period for example to edit step 3 in method 7 GET DEV METHOD 7 03 lt do gt DEV 7 03 IN 3 OUT O0t 12 0minT 35 C Or start from the beginning of the method and press step forward until the step you want to edit appears on the display Use the gt and 4 keys to move to the field you want to change Once the cursor rests under tne entry you want changed press CE for example to change the time in the above example DEV 7 03 IN 3 OUT 0 t 00 0min T 35 C Then enter the new value To insert a step or copy or delete a method or method step see Using the Keyboard where these keys are described Editing a running method To edit a running method you must first press DEV pause continue unless you only want to program or change the ala
99. r spans 10 000 daltons Gianzza E Righetti P G J Chromatography 193 1980 1 8 By kind permission of the authors and publisher PhastSystem User Manual 80 1320 15 Edition AK Description of the system 3 15 Non denatured proteins Relative abundance PhastGel Gradient 8 25 PhastGel Gradient 10 15 i t i Tha ey 100 300 500 700 900 Molecular weight x 10 Fig 10 The approximate molecular weight separation ranges of PhastGel gradient media are superimposed on a histogram showing the molecular weight distribution of native proteins The histogram is made up of data collected from 530 proteins Eachbar spans 10 000 daltons Gianazza E Righetti P G J Chromatography 193 1980 1 8 By kind permission of the authors and publisher PhastGel buffer strips PhastGel buffer strips are made of high quality agarose which has been countercharged and therefore has a low electroendosmosis Agarose IEF The agarose is melted together with buffer and then cast in the moulds The PhastGel buffer strip holder holds buffer strips in place on the gel Two buffer strips are used for each gel one at the cathode one at the anode The electrodes rest on the strips during electrophoresis and transfer current and voltage to the gel PhastGel buffer strips are individually sealed in airtight packages Once the buffer strips are removed from the package they must be used immediately PhastGel Blue R
100. r the measured temperature and adjusts the process time by this factor Therefore time is continuously integrated with a function of the measured temperature the Ct factors so that results will be reproducible regardless of the incoming solution s temperature The following example will help illustrate how temperature compensation works Example 1 A development method has been programmed with the following Ct curve DEV 2 Ct 5 30 40 50 C 0 5 1 3 2 0 2 6 The first step in the method has been programmed as follows DEV 2 01 IN l OUT 0t 12 0min T 50 C That is the process takes 12 0 minutes at 20 C for step 1 of method 2 DEV2 01 The actual temperature the step will be processed at T is 50 C Figure 2 illustrates how the programmed process time 1 is compensated when this step is run with solutions having different initial temperatures Plots 1 2 3 and 4 represent the same degree of development The run starting with solutions at 20 C 3 is processed faster than the run 2 starting with solutions at 4 C taken directly from the refrigerator The actual process time for both runs is approximately one half the programmed process time Solution temperature C 9 12 Time min Fig 31 PhastSystem automatically adjust the programmed process time for 20 C 1 for the methods run at 50 C starting with solutions at 4 C 2 at 20 C 3 and at 50 C 4 See example 1 for details PhastSystem User
101. real time When the display time reaches 10 minutes the step terminates and the next step begins The display time will be equal to real time when the temperature compensation function is not used that is when all Ct factors are set to 1 0 PhastSystem User Manual 80 1320 15 Edition AK 65 5 Operation 66 PhastSystem User Manual 80 1320 15 Edition AK Evaluation and presentation of data 6 6 Evaluation and presentation of data This chapter is divided into two parts preservation and evaluation The first part describes procedures for storing PhastGel media including how to dry and photograph gels The second part contains procedures for estimating the isoelectric point and molecular weight of proteins 6 1 Preservation Drying gels Use one of the following methods to dry your gels The method you choose will depend on how fast you need to dry your gels 1 Use an ordinary hair dryer to dry gels within minutes The warm air should be directed onto the plastic backing to avoid contaminating the gel with dust The gel should be dried immediately after development or uneven background can result This method is fast but higher background might result with Coomassie stained gels most likely due to Coomassie particles dissolving in the gel matrix if the air from the dryer is too warm 2 Place the gels on filter paper or a wire mesh Gels will dry within four to five hours Anchor edges of gradient gels to prevent them from curlin
102. rm Then select the method you want to edit in the DEV method file To change an entry in a running method Follow the directions above for editing a programmed method You can delete or insert 11 step in progress when you paused the run Running methods cannot be deleted Do not forget to continue the run when you finish editing your method press DEV pause continue PhastSystem User Manual 80 1320 15 Edition AK 55 5 Operation 56 5 6 Running a development method The procedure for running development methods comprises four steps making up the solutions connecting the bottles to the correct ports with the PVC tubing inserting the gels and pressing the start key The rest is automatic The procedure is described below Preparing the development unit You should always keep a fresh stock of solutions Filter solutions to keep the channels in the development unit clear and to avoid precipitation on the gel s We recommend that you label the bottles and the tubing use the yellow tubing markers to mark the tubes with their corresponding port number 1 Remove the caps on the cap set from the ports that you plan to use 2 Connect the ports 1 9 as required to the solution bottles with PVC tubing 3 Connect port 0 to waste Use an empty bottle 4 Check for kinks in the tubing Make sure the tubing is securely submerged in the solutions Important The chamber fills with approximately 70 ml of solution The bottles sho
103. ro Set the time t to 0 1 minute for each step DEV 9 01 IN 1 OUT 0 t 00 Imin T 00 C The out port will always empty to waste port 0 The chamber will empty immediately after it fills Running the cleaning method al a 3 Remove the cap set from the ports Cut the PVC tubing into 10 lengths one for each port Lead the tubes into a bottle containing at least 700 ml of distilled or de ionized water Lead tube 0 to waste use an empty bottle for waste Open the lid of the development chamber by pressing on the right end of the red bar Check that the lid gasket is secure Insert the level sensor shield into the upper position of the gel holder if it is not already there IMPORTANT The level sensor shield must remain in the chamber when running methods without gels otherwise the level sensor will be splashed by incoming solution causing it to give a false signal that the chamber is full 8 10 Close the lid and lock it simultaneously pressing on the lid and pressing in the red bar Press DEV start stop and enter the method number for example 9 START DEV 9 00 CLEANING lt do gt Press do to start the run Before using solutions other than water to clean the unit check the chemical resistance of the wetted parts to the chemical s you plan to use See chapter 3 Description of the system for more information about the chemical resistance of the wetted parts in the deve
104. ser Manual 80 1320 15 Edition AK The display Description of the system 3 The liquid crystal display prompts you for the correct series of entries when programming a method It also displays running conditions during a run and help messages which include messages for power failures and programming and system errors REAL CONDITION PROGRAM MODE SEP DEV SEP DEV real real method method condition condition file file SEP name insert method stand by temp SEP DEV pause pause continue continue SEP DEV help start start stop stop return Fig 11 Keyboard and display 3 5 The keyboard Numeric pad EG SEP ON Ga DEV ON step backward cursor 4 cursor The numeric pad on the right of the keyboard is used to enter parameters when programming a method or starting a separation or development run PhastSystem User Manual 80 1320 15 Edition AK 25 3 Description of the system Fig 12 Numeric pad CE The CE Clear Entry key is used to erase programmed entries The cursor must rest under the programmed entry to be erased The decimal or period key is used when entering a number containing a decimal point for example 2 4 volts and when entering a method number and method step for example 1 2 step 2 of method 1 This key must be pressed even though the decimal point is shown on the display m PROGRAM MODE SEP ete forward method file name step method
105. sis MEdId sssssmusenisusunsnsusurisisosiis 48 5 5 Programming development ProCcedUres esccssssssssesssseessssnneesseseessseees 52 5 6 Running a development ENO ajuikacaniiimieanaiamesoaaninn 56 S Canino method anere EA A 59 5 8 Temperature COMPENSATION sscccsssvsssssercnsssseceessonsssssecsssnsesseecssssesesesesnsnsssssereseess 61 6 Evaluation and presentation of data 6 1 Preservation Ln eeesesecssessesseesesssesscsssssecsussucsecsuesseesecsessucsussassuesarerecsuseseruccuesseceeneesussareasanees 67 6 2 EVICTION DO 69 7 Maintenance and trouble shooting Jal Separation andicontrol UNT ann aa 76 Te Development UNI cecvscictucesaiecveversieisterssinsisteistenssnnsitarectisislensioantasemementunenente 78 7 3 166172 12 19 012 9 9 aera tee eked nee enero enna nooner nee 80 TAC RECUCIN nns erre a r arae T AA EAE ETAR ETA REETA 80 PhastSystem User Manual 80 1320 15 Edition AK 5 Contents Help Message Reference Trouble Shooting Guide 8 Ordering information and technical data 8 1 Ordering ANTON CRON ca sssctieasinriindmannanenaaenen nen iammunaamaale 81 8 1 1 Gel media ANG OCCCSSOMES dacccetssscctecsscacntsctecdsedestestslavreatuasteies an na 81 8 1 2 Spare parts 8 2 Technical data 8 2 1 Separation ond control UNIT ss a ccisssssisciioancintts Hedidatieindanatincdiaanniioaitans 83 872 Development UNItascs cis wate nash Mon uh ss haoe As aaah ose i atau 84 823 OPIUM OLY OU eta sads cased bcc EE E E oben E E 84 8
106. sories This is a list of ordering information for gel media and accessories mentioned in this users manual Designation Quantity Code no PhastGel separation media PhastGel gradient 10 15 10 gels 17 0540 01 PhastGel gradient 8 25 10 gels 17 0542 01 PhastGel gradient 4 15 10 gels 17 0678 01 PhastGel homogeneous 7 5 10 gels 17 0622 01 PhastGel homogeneous 12 5 10 gels 17 0623 01 PhastGel homogeneous 20 10 gels 17 0624 01 PhastGel high density 10 gels 17 0679 01 PhastGel IEF 3 9 10 gels 17 0543 01 PhastGel IEF 4 6 5 10 gels 17 0544 01 PhastGel IEF 5 8 10 gels 17 0545 01 PhastGel chemicals PhastGel SDS buffer strips 20 strips 17 0516 01 PhastGel native buffer strips 20 strips 17 0517 01 PhastGel Blue R 40 tablets 17 0518 01 PhastGel silver kit for 10 20 gels 17 0617 01 PhastGel sample applicators PhastGel sample applicator 12 0 3 50 applicators 18 1614 01 PhastGel sample applicator 8 0 5 50 applicators 18 1617 01 PhastGel sample applicator 8 1 50 applicators 18 1618 01 PhastGel sample applicator 6 4 50 applicators 18 0012 29 Sample well stamp 1 18 0097 01 Molecular weight calibration kits HMW SDS high molecular weight in SDS 10 vials 17 0615 01 HMW High molecular weight 10 vials 17 0445 01 HMW Low molecular weight 10 vials 17 0446 01 PMW Peptide molecular weight 10 via 80 1129 83 pl calibration kits Broad pl range 10 vials 17 0471 01 Low pl range 10 vials 17 0742 01 High pl range 10 vials 17 0473 01
107. ssage will appear on the display The microprocessor will also detect programming errors or instrument malfunctions during operation In this case an alarm will sound running methods will be paused and a message will appear on the display telling you what is wrong These messages called help messages are listed by number in chapter 7 where you can find moreinformation about trouble shooting Separation compartment The separation compartment in the separation and control unit contains a separation bed with positions for two gels There are two alternate positions for each gel The vertical position with the tab at the front is the normal position The horizontal position with the tab to the left is for running the second dimension in electrophoretic titration curves PhastSystem User Manual 80 1320 15 Edition AK 13 3 Description of the system 14 The Peltier element automatically cools and heats the separation bed to the programmed temperature The programmable temperature range extends from 0 C to 70 C see cooling capacity page 14 The heat generated during electrophoresis is transferred to a large air cooled heat sink A standby temperature can be programmed to cool or heat the separation bed before methods are started This saves time since a method will not start until the bed temperature equals the programmed temperature for the first step in that method The electrode assembly contains two anodes and one cat
108. th the wells facing upwards 2 Choose the lane of holes that corresponds to the sample applicator you plan to use Place a piece of Parafilm with the protective cover facing upwards over the lane of holes 3 Run a pen or other hard object along the lane of wells to make depressions in the Parafilm 4 Remove the protective cover and place the Parafilm on a table so that the depressions can be filled with sample Sample well stamp Pap Parafilm Preparing sample wells Fig 16 Preparing sample wells 5 Fill the depressions with a volume of sample twice the applicator capillary volume For example if you use sample applicator 8 1 8 wells each 1 ul fill each well with 2 ul of sample Make sure there are no air bubbles in these samples as these will also be drawn up into the applicator capillaries 6 Mark the applicator for example left end or right end to avoid confusion later when inserting it into the applicator arm 7 Lower the applicator to the surface of the samples Break the surface of the samples and allow them to climb up into the applicator capillaries Avoid getting sample on the sides of the applicator PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Fig 17 Loading the applicator 8 Slide the loaded sample applicator into the appropriate slot on the sample applicator arm in the separation and control unit Do not press down on the applicator arm or samples may touch the gel surfa
109. the same type e g PhastGel IEF 3 9 See page 35 for further details 2 Press do to confirm 3 Enter the number of the method you plan to run START SEP METHOD 0 0 lt do gt The method always starts at the first step unless you enter a different step number after the period Once you enter the method number the method name if you gave your method a name will appear in parentheses beside the method number START SEP METHOD 3 0 IEF 3 9 lt do gt 4 Press do to confirm Monitoring the run If the separation bed temperature T is warmer or cooler than the programmed temperature T _ in the first step of the running method the display will show for example SEP 3 1 COOLING BED T 17 C T 15 C SEP 3 1 HEATING BED T 12 C T 15 C SET When T equals T the method will start and the running parameters will appear on the display for example SEP 3 1 450V 5 0mA 2 3W 15 C 03A Vh First the accumulated volthours AVh are shown i e the number of volthours that have elapsed since the beginning of the method run By pressing SEP real condition the number of elapsed volthours Vh for the running step is displayed Press SEP real condition to display AVh again PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Sample application 1 Load the applicator with sample as described earlier p XX 2 Press SEP pause continue The SEP ON LED will blink and an alarm will so
110. thods are available for programming Each separation method you program can contain two sample application instructions an extra alarm instruction and up to nine method steps The instructions and steps appear on the display one at a time A cursor rests under the field to be programmed For example in method 1 the first instruction will be SAMPLE APPL DOWN AT 1 0 0000 Vh At the cursor position enter the step number for sample application for example during step 2 By pressing gt the cursor will move to the next field SAMPLE APPL DOWN AT 1 2 0000 Vh At this cursor position enter the number of volthours that will elapse during step 2 before sample application By pressing step forward twice the first step of method 1 appears on the display SEP 1 1 0000V 00 0mA 0 0W 00 C 0000Vh Each method step finishes after a programmed number of volthours A method step can have up to 9999 volthours You must also program the following parameters for each step e Voltage in volts V 1 to 2000 V e Current in milliamperes mA 0 1 to 50 0 mA e Power in watts W 0 1 to 7 0 W e Separation bed temperature in C 0 to 70 C see cooling capacity page 14 Important Program methods for one gel Methods must always be programmed for one gel regardless of whether you plan to run one or two gels When you start a separation run a command will appear on the display where you must enter the number of gels yo
111. ting 76 6 Press step forward 9203 CALIBRATE DEV TEMP T XX C lt do gt 7 Press CE and enter the correct temperature 8 Press do to confirm 9203 DEV TEMP SENSOR CALIBRATED 9 Press SEP method file or any other functional key to exit this mode 7 1 Separation and control unit The maintenance required by the operator concerns the separation compartment The most frequent measure is cleaning the electrodes You may eventually have to replace the contact blocks and contact pins for the electrode assembly and the separation bed cover This is described below IMPORTANT For safety replace the separation compartment lid immediately if damaged Cleaning the electrodes The sample applicator arm and the electrode assembly constitute a unit which is fastened by two contact pins To clean the electrode assembly or to replace the contact pieces you must remove the unit Carefully pull the unit straight towards you be careful not to scratch the separation bed cover When you have pulled out the applicator arm and electrode assembly lay it down on a table and raise the applicator arm It will be easier to reassemble the unit if you don t dismantle it completely Pull out the contact blocks somewhat to free the electrode assembly Rinse the assembly in running water and let it dry Note Before you reinsert the electrode assembly check that all the electrodes lie on the same plane as the frame and
112. tion lid open During operation If a system problem occurs during operation the running method will be paused automatically an alarm will sound and one of the following messages will appear on the display 201 gt FAILED TO POSITION V3 TO EMPTY 202 gt FAILED TO POSITION V3 TO FILL 203 gt FAILED TO POSITION V3 TO NEUTRAL 204 gt FAILED TO POSITION V10 TO EMPTY 205 gt FAILED TO POSITION V10 TO FILL 206 gt FAILED TO POSITION V10 TO NEUTRAL 207 gt LEVEL SENSOR IN DEV CHAMBER NOT OK If codes 201 to 207 appear on the display call for service These messages appear when something is wrong with the valves V3 or V10 or the level sensor in the development unit 208 gt FAILED TO FILL CHAMBER SEE MANUAL This message will appear if the chamber cannot fill properly This message can appear even if the chamber is full In this case the level sensor can not sense liquid in the chamber Try the following suggestions to correct the problem e Check if the chamber is full or empty PhastSystem User Manual 80 1320 15 Edition AK 5 Help Message Reference Empty chamber or semi full chamber e Check bottles see if they have enough liquid in them and that the correct bottle is connected to the port e Check tubing see if there are any kinks or punctures that prevent liquid flow to the development chamber Check tat they are submerged in the liquid e Check the development lid see if it is closed tight or if the gasket is lo
113. ture Normally it takes 3 to 4 minutes to heat solutions to 50 C Once the programmed temperature is reached it is held constant within 2 C for the duration of the step Temperature compensation Temperature compensation is a programming option that is used for methods that contain very short steps or steps that are highly sensitive to temperature variations It automatically adjusts the programmed process time to compensate for the time required to heat incoming solutions to the programmed temperature Therefore solutions do not need to he pre heated before they enter the development chamber The development methods in the Development technique files chapter 9 do not require the temperature compensation function See page 61 for more information In the next section you will learn how to program development methods Each method has a temperature compensation instruction with default values set to 1 0 for example DEV 1 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 Leave these default values set to 1 0 unless you plan to use temperature compensation How to program methods A step by step instruction for programming development methods is given below Remember help messages can be accessed at any cursor position by pressing help return Selecting a method 1 Press DEV method file The method numbers that are free for programming are displayed in the parentheses GET DEV METHOD 0 00 FREE 123456789 2 Enter the number
114. u plan to run When you run two gels which must be the same type e g two PhastGel IEF 3 9 gels the current and power are automatically adjusted so that both gels run under the same conditions according to the programmed method The sum of current for both gels cannot exceed 50 0 mA and PhastSystem User Manual 80 1320 15 Edition AK 37 5 Operation 38 the sum of the power for both gels cannot exceed 7 0 W The following three examples illustrate this Example 1 The following step is programmed SEP 1 1 2000V 50 0mA 7 OW 15 C 0200Vh If one gel is run the limiting values are 2000 V 50 0 mA and 7 0 W which are the maximum values PhastSystem can deliver If two gels are run the limiting values for each gel are 2000 V 25 0 mA and 3 5 W Example 2 The following step is programmed SEP 1 1 2000V 12 5mA 3 5W 15 C 0200Vh If one gel is run the limiting values are 2000 V 12 5 mA and 3 5 W If two gels are run the limiting values for each gel are 2000 V 12 5 mA and 3 5 W Example 3 The following step is programmed SEP 1 1 2000V 30 0mA 5 0W 15 C 0200Vh If one gel is run the limiting values are 2000 V 30 0 mA and 5 0 W If two gels are run the limiting values are for each gel 2000 V 25 0 mA and 3 5 W Note The sum of the current for two gels and the sum of the power for two gels exceeds the maximum values Thus the limiting values for each gel in this example are the maximum available How to program a meth
115. uld be filled with at least 75 to 80 ml of solution to allow for the residual solution in the tubing 5 Open the lid of the development chamber by pressing on the right end of the red bar 6 Check that the chamber gasket on the lid is secure Inserting the gels 7 Remove one gel from the separation bed with a pair of forceps use the tab of the gel backing Be careful not to touch the gel surface with your fingers since fingerprints stain and cloud the protein band 8 Slide the gel gel surface down into the upper position of the gel holder Remove the other gel and slide it gel surface up into the lower position of the gel holder Note If you are developing only one gel slide it into the lower position gel surface up PhastSystem User Manual 80 1320 15 Edition AK Operation 5 Chamber gasket x Lower gel holder position Fig 28 Inserting the gel into the gel holder 9 Close the lid and lock it by simultaneously pressing down on the top of the lid and pushing in the red bar Jf G7 Fig 29 Closing the development chamber lid PhastSystem User Manual 80 1320 15 Edition AK 57 5 Operation 58 Starting the run 1 Press DEV start stop and enter the programmed method number START DEV METHOD 0 00 lt do gt 2 Once you enter the method number the method name if you gave your method a name will appear in parentheses beside the method number for example START D
116. umber of accumulated A volthours Vh which have elapsed during the run that is during steps 1 1 1 2 and 1 3 in the above example Press SEP real condition one more time to display the progress of the method step currently running in this example it is step 3 SEP 1 3 1500V 02 0mA 3 0W 12 C 0025Vh Press SEP real condition again to return to monitoring the progress of the entire method AVh PhastSystem User Manual 80 1320 15 Edition AK 31 3 Description of the system 32 PhastSystem User Manual 80 1320 15 Edition AK Installation 4 4 Installation IMPORTANT The following information must be read to install your PhastSystem instruments correctly 4 1 Unpacking CAUTION The system should be installed on a stable laboratory bench providing a suitable working area CAUTION To maintain correct ventilation the system requires an appropriate amount of free space Do not block the ventilation inlets or outlets on the system Unpack the equipment carefully and check the contents of the carton against the packing list Save the packing material and the carton in case PhastSystem must be returned Check the equipment for any visible signs of damage that may have occurred during shipment Removal of locking screw Remove the locking screw on the left of the underside of the development unit The air pump is mounted on a rubber support and fixed with this screw during shipment Save
117. und at 20 second intervals to remind you that the method is paused 3 Open the lid and slide the applicator into the appropriate slot cathode middle or anode in the sample applicator arm Repeat this for the second gel 4 Close the lid and press SEP pause continue The SEP ON LED will again show a steady light During the course of the run you may at any time start a development run for a finished gel or program another separation or development method To go back and check on the separation run in progress press SEP real condition Slots for applicators Fig 22 Inserting the applicators Stopping the run At the end of the method an alarm will sound for 15 seconds and will continue to sound at one minute intervals until the method is stopped The method will continue to run under the same conditions as the last step before an empty step in the method until the method is stopped 1 Stop the method by pressing SEP start stop PRESS lt do gt TO END SEPARATION METHOD 2 Press do to confirm The display will show the temperature of the separation bed and the accumulated volthours for the method just ended SEP OV 0 0mA 0 0W 15 C 500 A Vh 3 Proceed with development immediately after the method is stopped PhastSystem User Manual 80 1320 15 Edition AK 47 5 Operation 48 5 4 Running electrophoresis media The procedure for running PhastGel electrophoresis media is essentially the same for bot
118. ust lie perfectly flat against the bed The spare separation bed cover is self adhesive Peel back the paper backing along the anode side about 2 3 cm Put this end against the rear edge in the recess and fold it down carefully Press along this piece with your thumb to ensure good contact with the bed Peel back the paper backing another 2 3 cm and press along this piece with your thumb Be careful not to trap air bubbles between the bed and the bed cover If air bubbles are present you will have to try again with a new bed cover Continue peeling back the paper backing and smoothing out the bed cover until the recession is covered PhastSystem User Manual 80 1320 15 Edition AK TI 7 Maintenance and trouble shooting 78 Fig 38 Exploded view of the contact block 7 2 Development unit The maintenance required by the operator concerns the gasket in the lid the gasket in the 10 port valve and the tubing between the 10 port valve and the chamber The gasket in the lid should be replaced when visibly damaged when filling and emptying takes longer than usual or when filling and emptying does not function properly the chamber must be air tight for filling and emptying The other two items should be replaced if they start to leak Replacing the lid gasket Just pull the gasket off Be sure to turn the recess in the new gasket outwards Replacing the 10 port valve gasket Opening the valve 1 Start a development run any method
119. y installed and intact Connecting the units Connect the separation and control unit to the development unit with the communication cable code no 19 6005 02 Voltage selector ee communication cable To mains on off To Voltage mains selector Fig 15 PhastSystem controls rear PhastSystem User Manual 80 1320 15 Edition AK Installation 4 Mains connection WARNING Only use mains cables delivered or approved by GE Healthcare Plug the mains power cords 120 V or 220 V into the input marked MAINS on the rear panel of the units Plug the cords into the wall outlet grounded to earth Important Always disconnect these cords when servicing the instruments WARNING Do not block the rear panel of the system The mains power switch must always be easy to access 4 3 Turning the system on The system is turned on by pressing in the on off button on the rear panel of the separation and control unit The development unit is automatically activated when a development method is started Diagnostics Turn on the system and check that the diagnostics are successfully completed PhastSystem does a self diagnostic test every time it is turned on If an error is detected during the test a message will appear on the display and an alarm will sound Temperature sensors Both units have a temperature sensor one is under the separation bed and the other is on the underside of t
Download Pdf Manuals
Related Search
Related Contents
APRS event tracker setup Zanussi ZOB35301XK Epson Net 10/100 Base-TX External Print Server Product Brochure Hackers : Bâtisseurs depuis 1959 PROTRON STAR User Manual Muvit MUSCP0320 screen protector NTG Manuel d`utilisation EH Diplomat 3 22001 Swiss Top-Loading Washer Use and Care Guide Laveuse à Copyright © All rights reserved.
Failed to retrieve file