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Part 1 - Bascom Palmer Eye Institute

1. 5 Operation 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 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 47 5 Operation 48 7 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 Outer anode A raiona the buffer strip holder Fig 26 Positioning the buffer strip holder 8 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 9 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
2. 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 0V0 0mA 0 0W 15 C 500 A Vh 3 Proceed with development immediately after the method is stopped 45 5 Operation 5 4 Running The procedure for running PhastGel electrophoresis media is essentially electrophoresis the same for both native and SDS techniques The only operational difference is the use of PhastGel native or SDS buffer strips and sample media preparation The general procedure for running PhastGel electrophore sis 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 iF 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 Adjust the electrode assembly to the high position by pressing up on both red eccentric levers until they click into place Raise the electrode assembly to the vertical position Remove the IEF gel cover Wipe off the separation bed w
3. When trying to copy a method to a pro grammed 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 it 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 A NEW STEP DO lt pause gt When trying to insert a step in a running sepa ration or development method without first pressing SEP pause continue or DEV pause continue Help Message Reference 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 compensa tion Ct factor 0 0 or a Ct curve that de creases with increasing temperature The tem perature compensation curve instruction i a programming option If you
4. 1 Uneven electrode contact withthe 1 Before starting a run press along gels the electrodes to ensure good con 2 Dirty electrodes tact with the gel 2 Clean the electrodes after every Uneven iso pH lines The buffer capacity of the sample is too high Smiling bands 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 elec trodes page 2 Se also page 42 in the System Guide Dilute the sample or reduce the volume applied Trouble Shooting Guide Symptoms Probable Cause Solution s 1 Sample focused for insufficient 1 Apply the sample at different er number of volthours sites Note the Vh s for coal t a one 2 Gradient drift over focusing CEEE their pI 2 Stop the run sooner Find the p correct volthours for focusing as in 1 above 1 Enzyme is inactive at its pl 1 Try changing the pH of the gel i after the run in an appropriate Loss of enzyme aie memoved lt dumny acu buffer This might result in band activity 5 diffusion however 2 Add cofactors to the detection solution Sample is applied too close to its Try the other two application points isoelectric point Sample precipitates at the site of application Trouble Shooting Guide Symptoms Probable Cause Solution s PhastGel gradient and homogeneous media SDS buf
5. 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 A 5 Press step forward twice to display the name field for method 3 SEP 3 A 5 Operation Press gt or 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 Press do to enter this character into the name field SEP 3 L I Continue with steps 5 and 6 above to enter the rest of the char acters 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 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 instruc tion steps Programming sample application 10 11 12 13 Press step forward for the instruction SAMPLE APPL DOWN AT 3 0 0000 Vh 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 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
6. 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 the 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 gas ket 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 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 pre
7. 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 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 DEV 7 01 IN 1 QUT 0 t 100mm 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
8. We Palle Ge Tella ye a ae ee ie Wavy bands at or near the point of sample application 1 Field strength too high for sample 1 Decrease the field strength for the application sample application step e g use 220 V 2 5 mA for IFF and 400 V 2 Air bubbles in the sample 1 0 mA for native PAGE 2 Make sure the sample does not contain air bubbles Dotted or slashed bands Trouble Shooting Guide Symptoms Probable Cause Solution s PhastGel IEF media 1 Particulates in the sample 1 Centrifuge or filter samples 2 Poorly soluble protein that 2 Try applying the sample at a dif precipitates when applied to the ferent point Decrease the field gel strength for sample application 3 The sample has dried and aggre i 200 V and 2 5 mA or even gated in the applicators before ae being applied to the gel The 3 Load the samples just prior to the aggregates are left on the gel and sample application step espe Streaking leak protein during the run cially blood and serum samples 4 Dirty sample applicators 4 We recommend that sample ap S Satnple overloading plicators be used only once 5 Dilute the sample 1 The fixing solution was too old 1 Recycle the fixing solution no 2 The proteins in the gel were not more than 3 to 4 times fixed soon enough after the 2 Bands wills tart to diffuse im separation mediately after the method is stopped fix the proteins in the gel as soon as possible
9. 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 Parafilm is a registered trade mark of American Can Company 5 Operation 5 3 Running IEF media 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 surface 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
10. the actual temperature of the solution in the chamber is measured every second From the Ct curve PhastSystem obtains the Ct factor for 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 1 OUT s0 t 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 Tis 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
11. 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 The aim of this section is to prepare you for programming editing keyboard 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 be familiar with the display and the keyboard In this section the key board 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 quota tion 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 If 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 corre sponding LED will blink as a remember T
12. 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 5 Operation 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 es timated 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 pre heated 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 e
13. 19 6005 02 Voltage selector communication cable To mains Le Voltage selector Fig 15 PhastSystem controls rear 4 Installation 4 3 Turning the system on 4 4 Before use Mains connection 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 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 the 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 Before using the development unit we recommend that you run a cleaning method to remove dust accumulated during storage and shipment A cleaning
14. 20 C 94 000 PMW kit SDS 2 512 1 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 pl 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 pl for Coomassie staining or 3 ml for silver staining of suitable buffer for elec trophoresis 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 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 Prepare the sample proteins in the appropriate buffer as given above 3 Carry out electrophoresis according to the method given for native or SDS PAGE in the Separation Technique File 4 Develop the protein bands using one of the techniques in the Development Technique File 5 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
15. 75 volthours SAMPLE APPL DOWN AT 3 1 0075 Vh 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 from 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 sounds automatically at the end of the method 37 5 Operation 38 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 0000Vh 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 gt and enter the limiting power up to 7 0 W for the first step SEP
16. 99 pure to denature sample pro teins Streaki gt a cele SDS precipitates upon freezing eae 6 Dirty sample applicator warm samples to 20 C before loading sample applicators 5 Dilute the sample 6 We recommend that sample ap plicators be used only once 1 Frozen buffer strips Do not freeze at any time Frozen 2 Not good enough contact be strips lead to proteins only run tween buffer strips and elec ning half the gel more blurry and Tode a very low ending current Always check the contact and gently bend the electrodes down a little if necessary Proteins only runing half the gel 1 Touching buffer strips with Avoid touching the buffer strips fingers with anything when using sensi Background smear on the gel tive staining methods Wear gloves Trouble Shooting Guide Symptoms Probable Cause Solution s Development Coomassie staining PhastGel Blue R Stain particles on the gel surface A blue area in the center of the gel Weakly stained bands The coomassie stock solution or the final solution was too old and or unfiltered 2 Staining temperature is too low Cupric sulfate precipitates 4 Dirty tubing The gel in the lower position of the gel holder was inserted gel side down The gel becomes splashed by incoming solutions Old stain solutions or poorly filtered stain solutions Coomassie is not sensitive enough for the sample con
17. 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 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 toconfirm 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 51 5 Operation 52 Naming a method 4 Press name method DEV 1 A 5 Press step forward six times to display the name field for method 7 DEV7 L A 6 Press P or lt q 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 C 7 Press do to enter this character DEV 7 C C 8 Continue with steps
18. PhastGel IEF 4 6 5 PhastGel IEF 5 8 PhastGel chemicals PhastGel SDS buffer strips PhastGel native buffer strips PhastGel Blue R PhastGel silver kit PhastGel sample applicators PhastGel sample applicator 12 0 3 PhastGel sample applicator 8 0 5 PhastGel sample applicator 8 1 PhastGel sample applicator 6 4 Sample well stamp Molecular weight calibration kits HMW SDS high molecular weight in SDS HMW High molecular weight HMW Low molecular weight PMW Peptide molecular weight pl calibration kits Broad pl range Low pl range High pl range 17 0540 01 10 gels 17 0542 01 10 gels 17 0678 01 10 gels 17 0622 01 10 gels 17 0623 01 10 gels 17 0624 01 10 gels 17 0679 01 10 gels 17 0543 01 10 gels 17 0544 01 10 gels 17 0545 01 10 gels 17 0516 01 20 strips 17 0517 01 20 strips 17 0518 01 40 tablets 17 0617 01 for 10 20 gels 18 1614 01 50 applicators 18 1617 01 50 applicators 18 1618 01 50 applicators 18 0012 29 50 applicators 18 0097 01 1 17 0615 01 10 vials 17 0445 01 10 vials 17 0446 01 10 vials 80 1129 83 10 vial 17 0471 01 10 vials 17 0742 01 10 vials 17 0473 01 10 vials Patent pending 79 8 Ordering information and technical data 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 Code no Quantity Separation and control u
19. grammed 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 0 V 104 gt DEV PAUSE PROHIBITED IF DEV OFF When trying to pause a development run not started 105 gt SEP PAUSE PROHIBITED 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 O DOES NOT EXIST When trying to call up step 0 steps are num bered 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 subse quent steps 112 gt DESTINATION METHOD OCCUPIED
20. is full or empty Empty chamber or semi full chamber e Check bottles see if they have enough liquid in them and that the correct bottle is con nected to the port e Check tubing see if there are any kinks or punctures that prevent liquid flow to the de velopment chamber Check tat they are sub merged in the liquid e Check the development lid see if it is closed tight or if the gasket is loose or damaged Re place the gasket if it is damaged See page 78 of the manual e If you suspect an obstruction in the tube be tween 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 accord ing to the instructions given on page 78 of the manual If this valve leaks change the gasket To continue the run press DEV pause con tinue The method will continue by FILLING the chamber Help Message Reference 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 Presskeys
21. 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 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 21 3 Description of the system 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
22. 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 7 Maintenance and trouble shooting 7 Maintenance and trouble shooting In this chapter instructions are given for the maintenance of the instru ments parts that your service department can easily and quickly per form This chapter begins with instructions concerning both instru ments 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 mes sages 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 again 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 s
23. 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 0minT 45 C ENDING METHOD When the chamber is empty the display will show for example DEV t 5 0 min METHOD 7 DONE 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 until C appears in the parentheses and press do to enter it into the name field DEV 9 C C Follow step 4 to enter the rest of the characters 6 Press DEV method file again 57 5 Operation 58 Press 9 and do 8 Press step forward Leave these values set to 1 0 DEV9 Ct 5 30 40 50 C 1 0 1 0 1 0 1 0 9 Press step forward Leave the alarm instruction blank EXTRA ALARM TO SOUND AT 9 00 t 00 0min 10 Press step forwa
24. 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 reserved for 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 are using temperature compensation en ter the time required for this step at 20 C re gardless 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 how ever 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 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 pro
25. temp 23 C 0 20 40 60 80 100 Average run time Time min Fig 4 Separation bed temperature vs time The plots represent the following conditions 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 3 Description of the system 3 2 The development unit Fig 5 below shows the lowest separation bed temperature maintained within 1 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 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 30 For 2 gels 7W 25 20 Achievable cooling temperature C 5 7W e ei 4W 0 er ae 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 The visible parts of the development unit are a stainless steel chamber
26. the gel Buffer strips will protrude above the compartments by about 1 2 mm 10 Lower the electrode assembly so that the outer electrodes the cathode and the anode furthest from it rest evenly on the buffer strips 11 Gently press down along the top of the electrodes The electrodes must have complete and even contact with the buffer strips 12 Lower the sample applicator arm Sample application 1 Load the sample applicator s as described on page 39 2 Slide the loaded sample applicator s into the slot nearest the cathode 3 Close the separation compartment lid 5 Operation Sample applicators W A Fig 27 Inserting the applicators Starting the run 1 Press SEP start stop and enter the number of gels for this run NUMBEROFGELS 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
27. 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 3 PROGRAM MODE DEV step forward method file insert step backward cursor help q return cursor Fig 13 Programming keys 24 3 Descriptiaon of the system 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 P press to move to the next field of entry within a method step lt q press to move to the previous field of entry within a method step You can move the cursor rapidly through a method step by depress ing these keys longer than one second These keys also serve as step ping keys for selecting characters when naming a method step step forward press
28. the protein bands according to one of the development methods given in chapter 9 Develop ment Technique Files 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 Plot the known pl value of each pI 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 Measure the distance from the reference to the proteins of interest Use the pH profile of the gel to interpolate the pI 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 6 Evaluation and presentation of data 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 their 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 anhyd
29. to move to the next step in a forward method step step backward press to move to the previous step in a backward 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 PENA name method press to assign a name maximum of 10 method characters to a 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 5 Press name method Press step forward until the method number you want to name appears on the display 25 3 Description of the system 26 4 Press P or 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 ste
30. 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 0 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 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 15 3 Description of the system 16 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 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 chamb
31. zone Separation zone polyacrylamide polyester 0 175 mm thick 4 8 C Do not freeze 43 x 50 x 0 35 mm 5 T 3 C 37 mm approx 22 umol ml gel pH unit linear 43 x 50x 0 45 mm 6 T 3 C 13 mm 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 32 mm 0 112 M acetate 0 112 M Tris in both zones 6 4 43 x 50 x 0 45 mm 13 mm 32 mm 0 112 M acetate and 0 112 Tris in both zones 6 4 7 5 T 3 C 20 T 2 C 6 0 t 3 C 12 5 T 2 C 83 8 Ordering information and technical data PhastGel homogeneous 7 5 Stacking zone Separation zone PhastGel high density Stacking zone Separation zone PhastGel buffer strips Dimensions Volume Material Buffer system native strips SDS strips pH native strips SDS strips Storage PhastGel sample applicators 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 PhastGel sample applicator 12 0 3 sample wells well volume 12 approx 0 3 ul of sample PhastGel sample applicator 8 0 5 sample wells well volume 8 approx 0 5 ul of sample PhastGel sample applicator 8 1 sample wells well volume 8 approx 1 ul of sample Phast
32. 1 t 0 5minT 28 C PROCESSING P1 The in port tube number Pl 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 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 01t 10 5 min 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 5 Operation 5 7 Cleaning method 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 DEVt 0 0min 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 gt 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
33. 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 65 6 Evaluation and presentation of data 66 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 indicates depending on the meter type Thick black paper can be placed around the gel on the
34. 3 1 2000V 02 5mA 3 5W 00 C 0000Vh 19 Press P 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 0000V 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 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 run
35. 5 5 Programming development procedures 06 5 50 5 6 Running a development method c ccccceeees 54 5 7 Cleaning method vacisdsnsensaxtncenmanmounane 57 5 3 Temperaturec mpensati fi assenirnnnrrkxri 59 Evaluation and presentation of data ccssccccccssssseeeeees 65 Gd BECCA HON is acawiacvtresnsenenancnarenemnbiennteecandinsinwniens 65 62 AOU essens R 67 Maintenance and trouble shooting sses 73 7 1 Separation and control unit s issessessecsssevessveneverss 74 7 2 Developmentunit sssseeeeeessssssssseressssssssrereesss 76 73 Tro blesh otingsssennsounanicunssnnunua 78 Ordering information and technical data 1cccccceseeeees 79 Bal Orderinginformation 25 sicsinasenassncdemirewns 79 8 2 Techmeald ta sereins erne rere 81 Important user information Reading this entire manual is recommended for full understanding of the use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the instrument The lightning symbol within an equilateral AN triangle is intended to alert the user to the risk of exposure to high voltages The earth ground terminal symbol is used to mark a functional earth terminal Should you have any comments on this manual we will be pleased to receive them at GE H ealthcare Bio Sciences A B S 751 82 Uppsala Sw
36. 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 DEV7 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 the 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 example 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 0t 00 0min T 00 C 15 Press P and enter the port number the first solution will exit through enter 0 to 9 for example enter port 1 to recycle solution 1 DEV7 0 IN 1OUT 1t 00 0min T 00 C 5 Operation 16 Press P and enter the pr
37. BED T 12 C T 15 C 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 5 Operation the running step is displayed Press SEP real condition to display AVh again 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 sound 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 oe 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
38. F 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 26 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 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 number you want to name Then you must press gt or 4q to call up a character and do to en ter it into the name field See page 25 of the manual Separation field messages Press help return to display information a field marked out with the curs
39. F 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 I 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 35 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 Development field messages Press help 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 compen sation see page 61 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
40. Gel sample applicator 6 4 sample wells well volume Patent pending 84 6 approx 4 ul of sample PhastSystem Separation and Development Technique Files Application Notes and Technical Notes are now available at http Awww gehealthcare com lifesciences GE imagination at work 85 00 dV W1WSddN ML Ad NAGSMS NI GALNIud Yj GE imagination at work
41. 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 pro gram the following parameters for each step Voltage in volts V 1 to 2000 V Current in milliamperes mA 0 1 to 50 0 mA Power in watts W 0 1 to 7 0 W 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 you 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 the sum of the power for both gels cannot exceed 7 0 W The following three ex amples illustrate this 35 5 Operation 36 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 va
42. ach temperature The staining intensity can be measured visually or with a densitom eter Use an arbitrary scale for visual detection 4 Drawa line parallel to the x axis across each curve at an appropriate O D or staining intensity and read the time re quired for the reaction to reach this value at different tempera tures An example is shown in Figure 32 Comparing the process speed at different temperatures 50 C ane 40 C ei 5 5 30 C 20 C 5 C Band intensity O D 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 61 5 Operation 62 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 t
43. all comb like pieces have a series of capillary wells Samples are drawn into the capillaries and held there until the applicator is low ered 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 3 Description of thae system electrode assembly may take up two horizontal positions depending on the setting of the two eccentric levers The lower 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 posi tion is used for PhastGel electrophoresis media where the outer elec trodes 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 sample well ee gt gt stam p PhastGel separ
44. ation media eee ae ee PhastGel sample applicators r 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 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 13 3 Description of the system 14 For maximum reproducibility the d
45. bases alcohols hydrocarbons aldehydes and ketones Chamber lid PP strong acids in hot strong acids aromatic moderate con hydrocarbons and bleach centration in high concentration alcohols alde hydes and ketones 1These parts are illustrated on pages 16 17 and 79 Ethylene propylene copolymer and terpolymer Polyvinylidine fluoride 5Polypropylene or polypropene 3Polyvinyl chloride At present PhastGel media are available for four types of electrco phoretic 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 chemi 3 Description of the system cals are described below See Separation procedures and Development procedures for detailed instructions for using these produc
46. ble Shooting Guide Symptoms Probably Cause Solution s No bands but blue background KAR the gel edges Small cracks and rough uneven surface after drying Blue blotches near 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 corre sponding port number Label tubes with their corresponding port num bers using the yellow tubing mark ers Check the program against the bottle arrangement Use the plastic tab from the gel back ing to handle the gels Use forceps Use the lowest heat setting on the hair dryer Trouble Shooting Guide Symptoms Probably Cause Solution s PhastGel IEF media No or too little copper sulphate in the staining solution Dark area across the gel Old fix and wash solutions Dark background 1 Old fix and wash solutions the first two solutions This is usually visible only after the gel is dry 2 The coomassie concentration is too high Dark uneven back ground staining 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 aft
47. centration applied to the gel Not enough liquid in the deve lopment chamber during staining Too low temperature for staining 4 The wrong method from the devel opment method file was started by mistake The gel did not rotate in the solutions 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 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 Ensure out the tubing Do not allow coomassie solution to dry in the tubing Rinse out the tubing Do not allow coomassie solution to dry in the tubing The lower gel in the holder must face upwards Filter the stock solution stable for one month Try silver staining or concentrate the sample Make sure the bottles contain at least 80 ml of solution and that the tubes are completely sub merged in the solutions Check the temperature sensor calibration see chapter on Main tenance in the System Guide Check the method make sure it is the correct one and that it corre sponds to the bottle and port number arrangement Check that the gel holder rotates during processing If it does not call service The gels can be restained even if they are dry Run the stain and destain steps again Trou
48. ch 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 IEF4 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 pI measurement Instructions for pI measurement in PhastGel IEF media are given below 1 Ifyou know the approximate pl of the protein of interest select a suitable PhastGel IFF gel If it is unknown use PhastGel IEF 3 9 2 Usethe appropriate separation method for IEF described in Separation technique file No 100 chapter 9 67 6 Evaluation and presentation of data 68 Reconstitute one vial from the Low pI High pl or Broad pI calibration kit in 30 40 pl of distilled water for Coomassie stain ing and in 2 ml for silver staining Reconstituted kit proteins can be store frozen at 20 C 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 pI measurements they should focus at the same point in the gradient After electrophoresis develop
49. 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 message will appear on the display The microprocessor will also detect programming errors or instru ment 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 more information 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 tit
50. d 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 must 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 b
51. do not want tem perature compensation make sure all the Ct factors are set 1 0 for 5 C 30 C 40 C and 50 C See page 53 of the manual for more in formation 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 tempera ture greater than 50 C 123 gt SEPARATION LID IS NOT CLOSED When trying ti start or continue a separation run with the separation 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 Check if the chamber
52. e 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 5 Operation Fig 19 Bending the tab up 4 Place the gel on a hard surface 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 Remove any excess liquid with absorbent paper Note If only one gel is being run make sure that the empty gel area is dry Use a pair of forceps to gently lift and peel the plastic film from the gel surface Lower the electrode assembly Check that the inner anode nearest the cathode and the cathode have c
53. ectrophoresis 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 homo geneous gels are PhastGel homogeneous 7 5 PhastGel homogeneous 12 5 and PhastGel homogeneous 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 19 20 3 Description of the system 12 Relative abundance PhastGe IEF 5 8 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 dist
54. ed 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 gt DEV pause con tinue unless you only want to program or change the alarm 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 53 5 Operation 5 6 Running a 54 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
55. ed 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 75 7 Maintenance and trouble shooting Fig 38 Exploded view of the contact block The maintenance required by the operator concerns the gasket in the Tse Dey Sopmeni lid the gasket in the 10 port valve and the tubing between the 10 port unit 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 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 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 dis
56. eden GE H ealthcare Bio Sciences AB reserves the right to make changes in the specifications without prior notice Warranty and Liability GE H ealthcare Bio Sciences AB guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by GE H ealthcare Bio Sciences AB GE H ealthcare Bio Sciences AB shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Trade marks PhastSystem PhastTransfer and PhastGel are the exclusive trade marks of GE H ealthcare Bio Sciences AB In view of the risk of trade mark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trade mark status at least once in each article Copyright 1995 GE Healthcare Bio Sciences AB All rights reserved No part of this product may be reproduced stored in a retrieval system or transmit ted in any form by any means without permission in written form from the company 1 Introduction 1 Introduction PhastSystem consists of a separation and control unit a development unit
57. ensors 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 rela tively constant Alternatively use a thermocouple to measure the temperature of the separation bed and development chamber temperature sensor 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 73 6 Evaluation and presentation of data 5 Press do to confirm 9202 SEP TEMP SENSOR CALIBRATED 6 Press step forward 9203 CALIBRATE DEV TEMP T XX C lt do gt 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 The maintenance required by the operator concerns the separation compartment The most freque
58. 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 pr 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 continue 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 Guide lines 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 pl to the gel T
59. er 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 DEB Temperature sensor Uf aK lt gt Gel holder XN f SY gt TN A l P Development chamber Fig 6 Development chamber 3 Description of the system 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 swel
60. er drying Place the gels in the development chamber and start the destaining step 1 Recycle the fix and wash solu tions 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 Symptoms Probably Cause Solution s PhastGel electrophoresis Rehydrate the gel in glycerol acetic acid water according to Develop ment Technique Files Dry it again No or too little glycerol in the pre serving solution Store the dried gel in a plastic slide holder or cover the gel again with Gels curls and or craks after drying Protein Too much glycerol in the last preserving solution the protective film you removed prior to separation L Do not add more than 10 glycerol fade eae The gel was exposed to direct 2 Store the gel in a fairly dark place shee tonase sunlight for an extended period e g a notebook Silver staining The developer solution is too old 1 Use fresh developer Check the or it contains too much formalde concentration of the formal Dark yellow hyde a o o or brown tion is 0 04 o aqeous faaan d The ee step temperature is formaldehyde ie really 0 015 too high formaldehyde in solution The meinen te con 2 The optimal temperature for this ene a sensitive step is 30 3 The optimal concentration is 10 8 3 in solution Trouble Shooting Guide Symptoms P
61. er 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 development unit 5 Operation 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 pro grammed 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 f
62. ering specific techniques or application areas A technological extension of PhastSystem is PhastTransfer PhastTransfer PhastTransfer brings speed reproducibility and convenience to semi dry 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 1 Introduction Phastsystem Fig 1 PhastSystem consists of a separation and control unit a development unit PhastGel separation media accessories and a technical support package 2 Important safety information 2 1 Connection to the mains supply 2 2 Safety arrangements 2 Important safety Information 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 Se
63. ew in place will make 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 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 31 4 Installation 32 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 Check the voltage range of the mains electricity supply 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 correctly installed and intact Connecting the units Connect the separation and control unit to the development unit with the communication cable code no
64. fer strips were used instead of native buffer strips Extra bands with native PAGE The native buffer strips were used in stead 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 prop erly so that proteins migrate too close to one edge Curved bands on one or both sides of the gel 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 con tact 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 Trouble Shooting Guide Symptoms Probably Cause Solution s 1 Proteolysis of sample proteins 1 Prepare samples at low tempera Loss of protein bands appearance of extra bands Incomplete protein dissociation with SDS PAGE ture Try adding protease inhibi tors Heat samples in SDS buffer 10 mM Tris HCl 1 0 mM EDTA pH 8 0 and 2 5 SDS 5 mercaptoethanol at 100 C for at least 5 min 1 Poorly soluble proteins Apply the sample under low cur 2 Particulates in the sample rent e g 1 0 mA Increase the separation time Ss kapre GDS ithe ape 2 Centrifuge or filter the sample 4 Samples stored frozen with SDS and not warmed prior to electro phoresis Use analytical grade SDS
65. he 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 22 3 Description of the system The display 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 CI REAL CONDITION PROGRAM MODE DEV method file SEP DEV real real condition condition insert SEP stand by temp SEP DEV pause pause continue continue SEP DEV start start stop stop step forward step backward gt cursor 4 cursor SEP ON DEV ON Fig 11 Keyboard and display 3 5 The keyboard Numeric pad 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 23 3 Description of the system Fig 12 Numeric pad The CE Clear Entry key is used to erase programmed entries The cursor must rest under
66. he PhastGel sample applicator TC is a special applicator for electrophoretic titration curve analysis 39 5 Operation 40 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 with 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 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 pl fill each well with 2 pl of
67. he back of the development unit Help Message Reference 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 sec onds runnings methods will automatically continue from where they were stopped once power is returned 212 gt TEMP SENSOR NOT CALIBRATED Calibrate the temperature sensors according to the instructions given on page 75 of the manual 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 Trouble Shooting Guide Trouble Shooting Guide This guide lists the symptoms in illustrated form when possible of so
68. he 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 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 PhastGel 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 H ealthcare 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 The IN port the port the solution will enter through Ports 1 to 9 can be used port 0 is reserved for waste The OUT port the port the solution will exit through Ports 0 to 9 can be used The duration of the step t in minutes Each step can be up to 99 9 minutes The actual temperature T the step will be proce
69. he time that would be required for each method step at 20 C using the general 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 to move the cursor to the next position press CE and enter the Ct factor for 30 C for example 1 3 DEV 7 Ct 5 30 40 50 C 0 5 1 3 1 0 1 0 4 Press p 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 Operation 5 Press gt
70. 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 oe Place gel s in the separation bed 3 min development chamber 1 min Load PhastGel Select a programmed sample applicator s 3 min 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 gel s 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 i gt Specific detection via conventional methods e g zymograms auto radiography blotting The time intervals listed above will depend on the technique that is run 1 Introduction This users guide includes the following chapters Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Important safety info
71. ith 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 i 46 Place a drop of water or insulating fluid approximately 60 75 pl onto the middle of the gel area s outlined by the red lines on the separation bed 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 Use a waterproof pen to mark the underside of the gel for identification You might have to wipe the back of the gel first 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
72. l condition Press to monitor the progress of a i running development method condition 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 number 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 29 4 Installation 4 1 Unpacking 4 2 Cable connections 4 Installation Important The following information must be read to install your PhastSystem instruments correctly 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 the locking screw in case you should ever need to ship the unit Leaving the scr
73. l 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 power failures methods will remain in pause until you press SEP pause continue or DEV pause continue The display will show 3 Description of the system POWER FAILURE METHOD SET TO PAUSE Analarm will sound and the LED will blink to inform you about the power failure SEP SEP standby temp Press to cool or heat the Mer separation bed before starting a separation temp 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 SEP real condition Press to monitor the progress of a ans running separation method condition DEV DEV rea
74. 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 Panchromatic film works well This film is sensitive to red thus blue and dark bands become more
75. ls 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 17 3 Description of the system 3 3 PhastGel media 18 and chemicals Table 1 A general guide for the chemical resistance of the wetted parts in the development 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 Teflon most chemicals extreme conditions to chamber Tubing bottles PVC strong acids and hot acids ketones and to 10 port valve bases in moderate hydrocarbons concentration alcohols aldehydes and bleach Chamber gel stainless most chemicals long exposure to salt holder and temp steel solutions sensor Chamber lid gasket EPDM strong acids and hot acids and aromatic
76. lues 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 method 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
77. me 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 is divided into two major parts separation problems and development problems Separation Symptom Probable Cause 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 Solution s was not removed No bands on the gel 3 gel 1 The plastic film on the gel surface 2 No electrode contact with the gel 2 The sample was not applied to the 1 Remember to remove the film before starting a run Check the electrodes to make sure they are even Gently bend them down a little Always check the electrode contact with gel or buffer strips 3 Check that the sample applica to
78. 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 33 5 Operation 5 1 Programming separation procedures 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 Introduction Nine separation methods 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
79. ning 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 5 Operation 5 2 Sample application 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 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 step forward until the step you want to edit appears on the display Use the gt and keys to move to the field you want to change Once the cursor rests under the
80. nit Contact block cpl 18 3691 01 1 Contact pin 18 1661 01 2 Eccentric lever 18 1665 01 1 Sample applicator arm 18 1663 01 1 Separation bed cover 18 1671 01 1 Plunger adjustable 18 1019 67 2 Fuse 500 mA L 120 V model 19 8459 01 5 Fuse 250 mA L 220 V model 18 1000 68 2 PhastGel IEF gelcover 18 0083 01 2 PhastGel buffer strip holder 18 1668 01 2 PhastGel sample well stamp 18 0097 01 1 Development unit Gasket dev chamber 19 0048 01 1 Gasket 10 port valve 18 9482 01 1 Cap set 10 port valve 18 0072 01 1 Valve kit 10 port valve includes distributing plate and channel plate 18 1019 61 1 Tubing PVC 5 meters 19 0182 01 1 Tubing kit 10 port valve to chamber 18 0192 01 1 Fuse 175 mA L 120 model 18 1627 01 2 Fuse 80 mA L 220 v model 19 6236 01 5 Tube markers 0 9 5 of each 18 0180 01 50 Common items Fuse 800 mA L 220 V model 19 3085 01 5 Mains power cord 120 V 19 2447 01 1 Mains power cord 220 V 19 2448 01 1 Communication cable 19 6005 02 1 80 8 Ordering information and technical data 8 2 Technical data 8 2 1 Separation and control unit A list of the technical data for PhastSystem instruments is given below Dimensions Weight Keyboard Display LEDs Alarm Capacity Programs Separation Development Programmable parameters Separation Development Internal power supply Circuitry protection Voltage range Error Current range Error Power range Error Internal Vh integrat
81. nt measure is cleaning the electrodes control unit 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 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 74 7 Maintenance and trouble shooting Fig 37 The electrode assembly Replacing the contact pieces The contact blocks and pins should be replace when damage
82. ocess time for this step for example enter 10 5 minutes you must press although it is shown DEV7 01 IN 1 OUT 1t 10 5min T 00 C Note If you 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 gt and enter the actual temperature you want the step processed at for example 50 C DEV 7 01 IN OUT 1t 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 0 t 12 0min T 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 the entry you want chang
83. omplete and even contact with the gel surface Run your thumb gently along the top of the electrodes Lower the sample applicator arm and close the separation compartment lid Fig 20 Positioning a gel 43 5 Operation 44 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 NUMBEROFGELS 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 the same type e g PhastGel IEF 3 9 See page 35 for further details 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 pr 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
84. onnected to other CE labelled GE Healthcare Bio Sciences 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 95 100 120 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 Ordering information and technical data 8 2 4 PhastGel separation media and accessories A list of the technical data for PhastGel separation media and accesso ries is given below Common data Gel material Gel backing Storage PhastGel IEF media Dimensions Gel matrix Separation length 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
85. or When programming a separation method GET SET METHOD 0 0 FREE 123456789 8 gt MAX 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 14 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 mes sages 10 13 above Help Message Reference With the cursor in any of the V mA Q or C fields in a method step SEP 1 3 OOOOV 00 0MA 0 0W 00 C OOOO0Vh 15 gt MAX U 2000V I 25 0mA P 7 0W T 70 C These are the maximum values you can pro gram 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 be low SEP 1 3 OOOOV 00 0mA 0 0W 00 C OOOOVh 16 gt MAX VOLTHOURS IN A STEP 9999Vh When starting a separation run NUMBER OF GELS 0 lt do gt 17 gt I
86. or range Battery back up memory Separation compartment Electrodes number material width spacing Cooling heating Temperature Sample application Safety precautions 460 x 300 x 138 mm W x L x H 6 2 kg 31 tactile keys AO digit alphanumeric liquid crystal display 4 LEDs for status information An audible alarm sounds at the end of separation methods 1 or 2 gels 9 methods available for programming 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 titanium 42 mm 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 81 8 Ordering information and technical data 82 8 2 2 Develo
87. or 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 the 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 Curve 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 40 and 50 C 20 C is the reference temperature on which the curve is based 59 5 Operation 60 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
88. pment 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 Electricity requirements mains voltage mains frequency power Power disturbance Mains failure 300 x 300 x 138 mm W x Lx 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 O 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 steel 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 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 Bio Sciences AB except for alterations described in the user manual e used as stand alone unit or c
89. 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 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 should 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 5 Operation Lower gel holder position Af amp 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 Fig 29 Closing the development chamber lid 5S 5 Ope
90. 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 6 Evaluation and presentation of data 6 2 Evaluation 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 Procedures for measuring the isoelectric points and molecular weights of proteins using calibration proteins are described A brief discus sion about evaluating PhastGel media with PhastImage is presented at the end Isoelectric point measurement Isoelectric points pI 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 pI can be interpolated from the pH gradient profile Three pI calibration kits are available from GE H ealthcare Table 1 Ea
91. ps 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 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 for 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 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 3 Descri
92. ption of the system 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 delete 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 es steps 3 and 4 in separation method 1 press SEP method 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
93. rase 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 H ealthcare 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 69 6 Evaluation and presentation of data 70 Table 2 Molecular weight calibration kits available for native and SDS electrophoresis ug marker Storage number Marker MW 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
94. ration 56 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 DEV 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 0 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 0min T 22 C FILLING P1 It takes approximately 15 seconds to fill the chamber When the cham ber 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 0
95. ration curves 11 3 Description of the system 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 cathode 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 lt m i Sample applicator arm Plunger Eccentric lever vA Electrode assembly Separation bed Separation compartment Fig 2 Separation compartment Sample application Samples are applied to gels with PhastGel sample applicators These sm
96. rd 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 zero Set the time t to 0 1 minute for each step DEV 9 01 IN 1 OUT 0 t 00 lmin 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 1 Remove the cap set from the ports 2 Cut the PVC tubing into 10 lengths one for each port 3 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 5 Open the lid of the development chamber by pressing on the right end of the red bar 6 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 Close the lid and lock it simultaneously pressing on the lid and pressing in the red bar 9 Press DEV start stop and enter the method number for example 9 START DEV 9 00 CLEANING lt do gt 10 Press do to start the run Before using solutions other than wat
97. ribution of proteins in both denatured and non denatured form Denatured proteins 15 Relative abundance PhastGel Gradient 10 15 PhastGel Gradient 8 25 50 100 150 200 gt 300 Subunit molecular weight x 107 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 bar spans 10 000 daltons Gianzza E Righetti P G J Chromatography 193 1980 1 8 By kind permission of the authors and publisher 3 Description of the system a Non denatured proteins Relative abundance PhastGel Gradient 8 25 PhastGel Gradient 10 15 i i t Tha oy f 100 300 500 700 900 Molecular weight x 107 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 Each bar 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
98. rmation Description of the system introduces you to PhastSystem Installation tells you how to install PhastSystem 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 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 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 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 included 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 cov
99. robably Cause Solution s The gel turns yellow or brown upon drying Dark blotches around gel edges Very dark gel black or mirror effect Weakly stained bands Acetic acid wash ineffective in stop ping 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 solu tions 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 11 12 8 Ordering information and technical data 8 1 Ordering information 8 Ordering information and technical data 8 1 1 Gel media and accessories This is a list of ordering information for gel media and accessories mentioned in this users manual Designation Code no Quantity PhastGel separation media PhastGel gradient 10 15 PhastGel gradient 8 25 PhastGel gradient 4 15 PhastGel homogeneous 7 5 PhastGel homogeneous 12 5 PhastGel homogeneous 20 PhastGel high density PhastGel IEF 3 9
100. rona automated electrophoresis Phast System am 80 1320 15 Edition Al GE imagination at work 1 Contents Contents Introd cthion csssssscscisesstcsancsivosesashaserscensestionesstuiaseseiniaiues 5 Important safety information ccccecccccssesscccsssssseeseees 9 2 1 Connection to the mains supply jssscicsssasassssscasavssaties 9 22 Safety arrangements s s1sevsaadcensceassaoiaviatsaseiancsaansaesees 9 2 3 Safety precationis o 2c ccedesvevecsouvreeaecciceneecixastcss 10 Description of the system essssssssssssseessssseessseesssssesesssseeee 11 3 1 The separation and control unit lt cessssssseseneneees 11 3 2 The developmentunit ssssssssisrsssssisesisessssssssssessssss 15 3 2 PhastGel media and chemicals i ssccccssuscisaseseiecs 18 34 Using the keyboard saredesasasscadevnieaseasastsaceianssanereeas 22 Oa Whe hey Date reenen o aR E 23 Installation ssissisesissirseessssesssscssessrsssssssisssteesessssoessskesens 31 Al Unpack senn on unun RaR E ONOR 31 4 2 Cable COMM ECON ssisersncssrriy is 32 4 3 Turning the system on sssssssssseseesssssseeeesssssssssreee 33 4A Pelore USE sirri irori EENE 33 Oper tiOM svsininsssnisrunsveanesvassssanssieessatesasesasssannnnnioninonntons 35 5 1 Programming separation procedures cceeeeeees 35 52 Sampleapplicatioi icanansaknansisuemunweuws 39 5 3 Running IEF media sccwonsamncantonenswinesens 41 5 4 Running electrophoresis media c cccccceeeeeeees 46
101. rs gel cover and buffer strip holder are positioned correctly Disturbances at one or both of the edges Occasional empty lanes Excess water on the bed sur rounds and floods the gel Samples were applied too close to the gel s edge Sample list on the plastic when you put the sample applicator in the slot Remove excess water after posi tioning gels onto the separation bed Use only about 60 75 pl un der each gel Increase the temperature of the bed if the humidity in your lab is too high Make sure the gel is positioned correctly within the red vertical outline on the bed Take care you when you insert the sample applicator especially when using the buffer strip holder Trouble Shooting Guide Symptom Probable Cause Solution s Dirty electrodes Clean the electrodes with a wet lint free 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 CS PKS el SAREE COS EC SIEEN Wavy bands Air bubbles present between the gel Use about 60 75 pl of water to posi T and the bed tion the gels on the bed Remove all x air bubbles between the gel and the nea bed Lg Local band disturbance Too much salt in the sample Dilute or desalt the sample For IEE try another sample application position Pe Al Cee cepa
102. run If the separation bedtemperature T is warmer or cooler than the programmed temperature T r 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 TSET 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 real condition the number of elapsed volthours Vh for the step which is running is displayed Press SEP real condition to display A Vh again SET 49 5 Operation 5 5 Programming 50 development procedures 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 t
103. scan the gel with PhastImage 6 Measure the migration distance of the calibration proteins and calculate their R values R distance of the band from the origin distance from the origin to the reference point 6 Evaluation and presentation of data Use the furthest migrating calibration protein as the reference point With SDS PAGE use the tracking dye position as the reference point 7 Plot the R values of calibration kit proteins against the logarithms of their molecular weight 8 Calculate the R 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 71 6 Evaluation and presentation of data MW 200 000 100 000 40 000 20000 4 10000 0 2 0 4 0 6 0 8 Ri 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
104. ssed at the maximum temperature is 50 C The development chamber can only heat solutions 5 Operation Each method also contains a special programming option called tem perature compensation that works in conjunction with tempera ture 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 temperature 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 DEV1
105. t 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 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 For120 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 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 electrical hazard An alarm will sound until the lid is closed or until the run is paused 2 Important safety information 2 3 Safety The voltage supplied by PhastSystem is capable of delivering a lethal tions electric shock The numerous safety devices and circuits built into the precautio 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 separa
106. 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 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 41 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 th
107. 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 method and to activate the standby temperature 27 3 Description of the system 28 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 G9 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 SEP start stop Press to start a separation run Press start again to end the run stop DEV DEV start stop Press to start a development run Press Sari 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 wil
108. 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 firstand discon nect the tubing from the valve Remove the black cover plate for the tubing using a Philips screw driver 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 Help message reference shooting 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 78 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 refer ences to further information concerning the help message In some instances suggestions are 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 OF
109. tion 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 10 3 Description of the system 3 1 The separation and control unit 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 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 work together to form a system for fast electrophoresis PhastSystem 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
110. tributor 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 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 76 7 Maintenance and trouble shooting 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 10 Re connect mains power cable Press gt DEV pause continue and then DEV start stop If valve leaks open and check that all parts are correctly mounted Note If the valve still leaks you may have to replace the distributing and channel plates also Fixing plate Channel plate Distributing plate VA Gasket 3 Z Distributor f Pressure i plate N The 10 port valve Fig 39 The 10 port valve Start here The 10 port valve to chamber tube Fig 40 The 10 port valve to chamber tube OE 7 Maintenance and trouble shooting Replacing
111. ts 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 of PhastGel IEF media with respect to the pI 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 H ealthcare molecular weight calibration kits See Evaluation and presentation of data chapter 6 for instructions Seven different gels for el
112. uration 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 capacity 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 IFF 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 20 For 2 gels 18 16 Cooling bed temperature C 3 Native and SDS PAGE ambient temp 38 C 12 2 IEF ambient temp 28 C H 1 IEF ambient
113. vious 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 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 gt EMPTYING step start the step again as described above To change the 10 port gasket If the chamber will not fill despite the above mea sures try changing the 10 port gasket Follow the directions on page 78 of the manual To continue the run press DEV pause con tinue The method will continue by FILLING the chamber 210 gt POWER FAILURE IN DEV UNIT Check that the development unit is plugged into the wall outlet Check that the development unit is prop erly connected to the separation and control unit via the communication cable Check the fuses in t
114. will elapse faster than 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 63 64 6 Evaluation and presentation of data 6 1 Preservation 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 includ ing how to dry and photograph gels The second part contains proce dures for estimating the isoelectric point and molecular weight of proteins 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 Usean ordinary hair dryer to dry gels within minutes The warm air should be directed onto the plastic backing to avoid contami nating 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 curling If your gels curl after drying or storage soak them in 7

Part 1 - Bascom Palmer Eye Institute

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