Instrument User Manual
Contents
1. PRODUCT CODE DESCRIPTION PRO 0012 EWS 1 Electrophoresis WorkStation PRO 0010 P Focus IEF system PRO 0050 IEF strip tray 6 places PRO 0052 IEF strip tray 12 places PRO 0060 IEF eguilibration tray for 8cm lenght strip 14 places PRO 0062 IEF eguilibration tray for 24cm lenght strip 6 places PRO 0064 IEF eguilibration tray for 45cm lenght strip 6 places PRO 0070 IEF rehydration tray for 8cm lenght strip 14 places PRO 0072 IEF rehydration tray for 24cm lenght strip 6 places PRO 0074 IEF rehydration tray for 45cm lenght strip 6 places PRO 0078 IEF Precut rectangular paper bridges 50pz PRO 0080 Cover Fluid PRO 47712 IPG strip 4 7 7cm 1 5mm thickness 12 strips PRO 471112 IPG strip 4 7 11cm 1 5mm thickness 12 strips PRO 472412 IPG strip 4 7 24cm 1 5mm thickness 12 strips PRO 310712 IPG strip 3 10 7cm 1 5mm thickness 12 strips PRO 310NL712 IPG strip 3 10 NL 7cm 1 5mm thickness 12 strips PRO 610712 IPG strip 6 10 7cm 1 5mm thickness 12 strips PRO 58712 IPG strip 5 8 7cm 1 5mm thickness 12 strips PRO 36712 IPG strip 3 6 7cm 1 5mm thickness 12 strips PRO 3101112 IPG strip 3 10 11cm 1 5mm thickness 12 strips PRO 310NL1112 IPG strip 3 10 NL 11cm 1 5mm thickness 12 strips PRO 6101112 IPG strip 6 10 11cm 1 5mm thickness 12 strips PRO 581112 IPG strip 5 8 11cm 1 5mm thickness 12 strips PRO 361112 IPG s2. P FOCUS 4 JELETTROFOR Scientific Instruments amp TD imenstonal ti l Scientific Instruments f MEN LO na Index UE a 0 RYFAIN EY FFF FFR rr 2a2 4 2 dae VOM OL CES C11 01 In SYR NN TT E RR ll RYDYN 5 3 Isoelectric focusing applications 6 SEED 8 Ke 82 6o O ee ane FRON EN O r en A NF EF dos 8 3 2 Rehydrating and loading IPG strips 9 4 Low voltage electrophoresis applications for EWS 1 only 9 eeeeeuuuen 14 5 a Control SOltw AS AAA a k ka ea W ya G 15 5 1 General informations about cursor keys 16 5 2 Plate connectors Ior EWOS T only ea OR GDG Rd Wan neke a n k ban Ann 16 39 HOW WO t m on the Sy SCC TN RY Y YO AW0NR da eee FERN FN RWYF YY ENFAWR 17 JA How touse Mes ystle L L FYR a ea a ee ae mz mz zm 17 SS a n ME TU EWCH rrr r rr rrr yy rer r rr 17 ES NS __Ly e 17 sS IE o SAS a en nee ee to FFF 18 D 02 PO SOS CD 2 FR EOW mm 22 OS D E T FRYN YN AER YNYR 25 5 7 Low voltage power supply for EWS 1 only kk 26 A Mh FOIA Der PP ak goooa r r w w wa aa w wrX amp bb r reeg g 0_ __m_m_DD 26 Didi ka TOES Ne MINES PRYFYN ee ren CF REF EFA BB te in 26 Ss MS RABE 10 PAUSE a AN Y PF FY YF EN E WY TR ND FYNED RF NF 29 IS POr a 66 ACCS EYN RR FR CAER CREFFT EU NF RNA RHYN FC Y REF 29 6 Eu n 0 8 8 SYN ets b Kenede eb aie FY emel n n b FF 31 RCD E aes negate ce E T E sede memies eta aenee 32 e EE E LE i
3. Electrophoresis 26 16 3185 90 e Xiao Z Conrads TP Lucas DA Janini GM Schaefer CF Buetow KH Issag HJ Veenstra TD 2004 Direct ampholyte free liquid phase isoelectric peptide focusing application to the human serum proteome Electrophoresis 25 1 128 33 36 2 SP re T Dimensional 37 4 ELETTROFOR Ty p amp a Scientific Instruments L m E nm 4 L O nm a
4. completely rehydrated not properly closed check the rehydration times and volumes check that the lid 1s correctly closed The voltage 1s not The salt concentration of the Desalt the sample replace increasing during the IEF sample is too high the paper bridges The maximum voltage is The programmed voltage is We recommend to not reached very slowly too high for the strip length exceed a voltage gradient of the salt concentration of the 330 V cm desalt the sample sample is too high replace the paper bridges after 2 hours of IEF Burning of IPG strip The current limit is too high We recommend a current the strip has dried out paper limit of 50 uA per strip bridges are too wet wrong make sure that the strip is paper bridge solutions completely covered by the Cover Fluid make sure that the paper bridges are only wet not saturated or dripping use deionized water only Formation of urea crystals Temperature of cooling Set the temperature at 20 C on the IPG gel surface plate too low IPG strip turn white The strip has dried out Make sure that the strip is during IEF completely covered by the Cover Fluid Current does not drop Wrong orientation of the Check that the strip during the IEF initial strip acidic end towards orientation is correct desalt stage cathode salt concentration the sample replace the paper of the sample 1s too high bridges 31 ib 0 EF Td D
5. is a type of zone electrophoresis that takes advantage of the fact that overall charge on a protein is a function of the pH of its surroundings Proteins carry charged groups on their surface Each of these functional groups has a pK which corresponds to the pH at which half of the members of that group are protonated Above 6 r SLman loun or below the pK that group can be considered respectively fully protonated or deprotonated Thus as the pH changes the net charge on a protein s surface will change The isoelectric point pl is the pH at which the net charge of the protein is zero With the presence of a pH gradient in the IEF method the protein will migrate to the position where its charge is zero a protein with a positive net charge will migrate toward the cathode until it meets its pl while a protein with a negative net charge will migrate toward the anode until it meets its pl The matrix used in this process has a high porosity to eliminate any sieving effect which would cause differing migration rates for proteins of different sizes If the protein diffuses away from its pl it will regain its charge and migrate back A reproducible pH gradient is crucial for successful IEF In order to overcome the limitations of carrier ampholyte generated pH gradients e g the so called cathodic drift and plateau phenomenon immobilized pH gradients IPG were developed in 1982 IPGs are based on the bifunctional Immob
6. this section you can choose and run a program e Before and during the execution of a program you can change the value of some parameters This changes will be operative only during the current session because data is not stored on memory e The screen shows the name of the program as it was set in the SETUP PROGRAM section for the specific program selected e The REHYDRATION PHASE can be included or excluded from the current program in which case you can change the duration time e Before running selected program you must set the strip number that will be used STRIP NO for the current session e Each step include a voltage value VOLT a duration time TIME an operative mode MODE and a remaining time REMAIN TIME which indicate how much time is left to ending the current STEP e For each program the system will calculates the total duration under the TIME column and the total remaining time REMAIN below the column TIME These are the two TOTAL TIME and are composed with time associated to the STEP and the rehydration phase 1f included PROGRAM SELECTION Move the cursor key on the program number NR PROG and use the and to select the desired number program REHYDRATION PARAMETER e Before the execution of a program Alongside REHYDRATION total duration of rehydration phase is displayed If the values of hours and minutes are different from zero this means that the program is running with the rehydration
7. time egual to the previous time set up If the values of hours and minutes are 00 00 the rehydration phase is not included and protocol will begin with the first non zero STEP 18 L IELEITSTROFOR Tir f Scenic Tm a au K Z f L m amp nm 4 L O nm a e During the execution of a program In this case alongside REHYDRATION will be displayed the value of remaining time If rehydration phase 1s included and 1s currently running you have the possibility to change the remaining time If this time is set up at 00 00 it means that you want to end prematurely the rehydration phase this change is 1mplemented after few seconds Instead If the remaining time is increased this is only possible 1f the total duration of rehydration phase does not exceed the maximum allowed Length Total Elapsed Time Time Remaining lt 99 59 STRIP NUMBER AND POWER LIMITATION e Before starting a program you must set the strip number STRIP N You can enter values between 1 and 12 e When setting this parameter a check to the maximum power required is automated made in relation to the maximum current per strip set in the SETUP section It would be possible that the maximum number of strip number set is less than 12 PARAMETERS VIEWING AND CHANGING e On entering this section you will see the table containing STEP to 5 e To see next 6 10 STEP move the cursor in one step 5 parameters and press DOWN or mov
8. L TYPE select the control mode When this parameter is changed the value and measuring unit are updated automatically During system working 1s ever performed voltage control to the connectors and the load current order to not exceed the max output values allowed by the system VOLT voltage control is selected and active the system acts on the value of output voltage in order to ensure the voltage value set as a parameter CURRENT current control is selected and active the system acts on the output voltage to achieve and maintain a current output egual to the desired value POWER power control is selected and active the system acts on the value of output voltage and monitoring the load current trying to achieve and ensure the power value set as a parameter e VALUE SET Based on the type of control chosen you can set its parameter number within the specified range see the next chapter on electrical characteristics and general e TIME time management of the device If time value is 00 00 there is no countdown set In this case all the operations must be stopped manually until the user decides to interrupt the session by pressing STOP button If the value time is different than 00 00 all the operations will stop automatically once timer finish the countdown and supply will turn off 21 F ELET GROFOR TN p f 2 A acien TIF lc ns Trumen ts f L nh nm 4 L O nm a 5 7 3 Start a
9. OMPLETED and LED will become red e By Pressing one of the two selection keys you will see the protocol STATISTICS Here the system will report the following data Identification program number Name of the program Rehydration phase total lenght Number of Strip VOL T hour for each step Total time for each step The sum of the values VOLT7hour of all the steps that are included in the protocol The voltage applied during rehydration phase 1s not considered The total time of all STEP To viewing over STEP 1 5 and STEP 6 10 press UP and DOWN keys To exit from STATISTICS and return to the Main Menu press one of two push buttons ENTER or ENTER 21 az EG TO imensional j 5 6 2 Program Setup In this section you can define up to 10 different programs All data and parameters set written in this section are saved in memory MAIN FEATURES OF PROGRAMS e Each program is characterized by an identity number PROG NR from 1 to 10 e It is possible to associate a name to each program maximum length 16 characters e Each program contains the data of a protocol with a maximum of 10 STEP it is possible to choose whether the protocol should be preceded or not by the rehydration phase according to the features planned in the SETUP e Each step is composed by a voltage VOLT a duration TIME and an operative mode MODE values e The total duration TOTAL TIME is calculated how the sum of a
10. OP PAUSE F key RIGHT gt L red LED if machine is not working 15 ELE TTROFOR TD imensional 5 1 General informations about cursor keys e The cursor keys UP DOWN LEFT and RIGHT are used to navigate through the display The current cursor position is indicated by highlighting text e Increase selection ENTER and decrease selection ENTER key are used to confirm for example if you want to exit from an application and the cursor was placed on EXIT Also is used to change parameters In most cases to guickly change a parameter simply hold down the button e The START and STOP PAUSE keys are active only in OPERATING STAGE respectively to start or resume execution of a protocol or to stop 1t temporarily or permanently To ensure the execution of these actions you must be press button for a_ longer time that the normal 5 2 Plate connectors for EWS 1 only The position of high and low voltage connectors is schematically reported in figure 10 Connectors to the high voltage module IEF SYSTEM Connectors to the low voltage POWER SUPPLY for EWS 1 only Figure 10 16 EP 7 ZAN Td O imensional 5 3 How to turn on the system Make sure that the power cord is correctly plugged 100 250 50 60Hz Turning on the mains power switch located on the side of the EWS 1 and of the P Focus activates a self diagnostic program that runs for approximately 10 seconds If any failure 1s de
11. RE protocol temperature C CURRENT STRIP max current per strip 5 7 Low voltage power supply for EWS 1 only 5 7 1 Initial informations Using the low voltage power supply thermostatic control of the plate is not provided Moreover 1s not necessary to use the protective cover All setting parameters are stored in the system memory When you turn on the system when you log on to the POWER SUPPLY application the parameters set during the last session are loaded The bottom row of the display is the status bar and shows the current status of the device POWER SUPPLY STATUS 1 STOP 2 PAUSE 3 RUN During stop and pause the power is off red LED When RUN the power is on green LED illuminated 5 7 2 Parameters Setting Setting of parameters is allowed only when the power supply is not working STOP With the cursor keys and the and it 1s possible to choose and modify and one of the four parameters and manage the operation of the power supply 26 A EF TIF Dimensional e MINIMUM CURRENT CONTROL control over the minimum current output NO no control over the current minimum is made YES a control is made on the minimum output current During protocol execution if current results below the minimum threshold see next chapter on electrical characteristics and general the power is turned off and an error message will appear together with a warning acoustic sound e CONTRO
12. c focusing as a first dimension separation in shotgun proteomics J Biomol Tech 16 3 181 9 e Eriksson H Lengqvist J Hedlund J Uhlen K Orre LM Bjellqvist B Persson B Lehtio J Jakobsson PJ 2008 Quantitative membrane proteomics applying narrow range peptide isoelectric focusing for studies of small cell lung cancer resistance mechanisms Proteomics 8 15 3008 3018 e Fraterman S Zeiger U Khurana TS Rubinstein NA Wilm M 2007 Combination of peptide OFFGEL fractionation and label free quantitation facilitated proteomics profiling of extraocular muscle Proteomics 7 18 3404 3416 e Geiser L Vaezzadeh AR Deshusses JM Hochstrasser DF 2011 Shotgun proteomics a qualitative approach applying isoelectric focusing on immobilized pH gradient and LC MS MS Methods Mol Biol 681 449 58 e Heller M Michel PE Morier P Crettaz D Wenz C Tissot JD Reymond F Rossier JS 2005 Two stage Off Gel isoelectric focusing protein followed by peptide fractionation and application to proteome analysis of human plasma Electrophoresis 26 6 1174 1188 e Millioni R Miuzzo M Puricelli L Iori E Sbrignadello S Dosselli R Cecconi D Tessari P Righetti PG 2010 Improved instrumentation for large size two dimensional protein maps Electrophoresis 31 23 24 3863 6 e Poznanovic S Schwall G Zengerling H Cahill MA 2005 Isoelectric focusing in serial immobilized pH gradient gels to improve protein separation in proteomic analysis
13. e to EXIT and press the UP button e In addition during the execution of a protocol the cursor can be moved only within the range that goes from STEP or rehydration up to the first zero STEP e Changing the STEP parameters if protocol is not running for each step you can change the voltage VOLT and if this is different from zero the values of step duration TIME and the operative mode MODE e During the execution of a protocol is possible to change the voltage VOLT and if this is different from zero the remaining time REMAIN TIME PROTOCOL EXECUTION e To start a program press START button for a few seconds red LED will turn off and green LED will lights up 19 az EG TO imensional j e Two flashing arrows indicate the exact point of protocol execution e Throughout the execution of the program lid condition is constantly monitored and when open an intermittent beep sound will be audible and the message NOT LATCHED LID will be displayed In this situation voltage on strip is cut off and the plate temperature is no longer controlled To resume the current program running you should close the lid and press START button e When a protocol is running the display shows alternately a row containing the program identification number and name a row that shows the instantaneous values of voltage VOLT and current per strip CURRENT and a line which shows the cumulative value of volts time al
14. ed protein sample is possible in a freezer at 80 C preferred Repeated freezing and thawing of the sample must be avoided It is recommended to make aliquots of the sample and thaw only once L IELEITSTROFOR Tir f Scenic Tm a au K Z f L m amp nm 4 L O nm a e due to the high dynamic range and diversity of any proteome it is highly recommended to perform a pre fractionation step to reduce the complexity of the sample Over the past few years IEF has gained increasingly interest also as a peptide fractionation method Eriksson H et al 2008 Fraterman S et al 2007 Cargile BJ et al 2004 Cargile BJ et al 2005 Geiser L et al 2011 Heller M et al 2005 In fact apart from being an excellent tool for separating complex mixture of peptides peptide IEF has high reproducibility is compatible with other fractionation technigues for multistep protocols and can provide the additional information of experimental pl that can aid in the protein and post translational modification identifications In this approach the IPG gel strip is cut into sections and then the focused peptides can be extracted and analyzed from all the gel sections Cargile BJ et al 2004 One advantage in working with peptides is that peptides possess greater solubility than proteins Xiao et al 2004 demonstrated the efficacy of ampholyte free peptide autofocusing avoiding sample losses due to ampholyte removal strategies The most used sol
15. ee r rw yy 33 DESI AAT OF Fa 3 NF NNRRFFN E mr r2 34 MY ING Pe GCC FE YF YNYR FE AEE EF FYRFYFYR 36 3 O ELETTROFOR TD f CAK ZAZ kZXKI imenstona 1 Safety information WARNING The Warning sign highlights an instruction that must be strictly followed In order to avoid personal injury Be sure not to proceed until the instructions are clearly understood and all stated conditions are met The Electrophoretic WorkStation EWS 1 has been tested and complies with the IEC 61010 1 EN 61010 1 Electrical safety standard Extreme caution should be exercised when operate because this instrument can develop higher voltage and current sufficient to produce a lethal shock Read this entire manual before using the instrument and use it only according to the manufacturer s instructions Before using to make sure that e The instrument must always be used with the earth lead of the power cord correctly grounded to earth at the mains outlet e To permit sufficient cooling ensure that the vents in the over and under of the instruments are not covered e To use the instrument only indoor not extreme humidity above 95 Avoid condensation by letting the unit equilibrate to ambient temperature when taking the power supply from a colder to a warmer environment e Keep the instrument as dry and clean as possible Wipe regularly with a soft damp cloth e Let the power supply dry co
16. gure 6 Moistened prior to use with deionized water these paper bridges absorb excess water salts and proteins with pI values that lie outside the pH range of the IPG strip Transfer the IPG strips from the rehydration to the IEF tray The IPG strip acrylamide must be in contact with the paper bridges The acidic end of the IPG gel strips must face towards the anode Figure 6 Positioning the paper bridge the electrode and the IPG strip These operations must be done at both the ends of the strip Remove excess water from the paper bridges by blotting with tissue paper paper must be only wet not saturated or dripping 12 Sy ELETTROFO A TD imensional e The sliding electrodes are fully adjustable to suit the length of strips Since it 1s very difficult to produce more than 24 cm long pH gradients shorter IPG strips of appropriate pH ranges can be positioned end on end Poznanovic S et al 2005 During IEF proteins efficiently migrate from one IPG to another by traversing buffer filled porous bridges between the serial IPGs A variety of materials can function as bridges including paper or polyacrylamide gels or even the same IPG strips as shown in figure 7 Figure 7 A schematized example to show the loading of two A and three B IPG strips The plastic backing film is indicated by a heavy border on the IPG strips Arrows indicate the position of the electrodes The small blue rectangles indicate the paper wic
17. igure 3 Remove the protective plastic 2 from the IPG strip 1 10 V ELETTROFOR Scientific Imstruments TD LMENS LO n al e Lay down the IPG strips gel side down into the rehydration solution Wet the strip by sliding it back and forth along the tray channel Remove any large air bubbles For typical composition of rehydration solution see paragraph 10 e Completely cover the IPG strips with the Cover Fluid and allow the strips to rehydrate The Cover Fluid is used to minimize evaporation and urea crystallization A minimum of 10 hours is required for rehydration overnight is recommended Alternatively the rehydration can be programmed as the first step of the IEF protocol This is convenient if temperature control during rehydration is a concern or if a low voltage is applied during rehydration e Wash the IEF tray extensively with double distilled water to remove residual proteins The tray must be completely dry before use Figure 4 IEF tray for 42 cm long IPG strips or for shorter IPG strips of appropriate pH ranges positioned end on end e Put the IEF tray on the cooling plate and connect the electrodes to the high voltage module figure 5 step 1 4 11 DZ TD imensional Figure 5 e At the bottom of the teeth of the sliding electrodes platinum wires provide electrical contact to the small rectangular paper bridges that must be placed at both the anodal and cathodal ends of the rehydrated IPG strips fi
18. ilines reagents which are acrylamide derivatives Their general structure is CH gt CH CO NH R where the group R contains either an amino or a carboxyl or group and forms a series of buffers with different pK values between 1 0 and 13 Since the reactive end is co polymerized with the acrylamide matrix the pH gradients are stable and reproducible also during extended IEF runs The preparation of IPG strips on a plastic backing offer an optimal solution for convenient handling The strip length depends on the size of the second dimension gels to be used with longer strips and larger gels providing higher sample capacity and resolution The main problem to further increase the resolution of IPG strip 1s the technical difficulty associated with establishing reproducible density gradients over distances longer than 24 cm This issue could be avoided by using two or three strips the number depending on the length of individual strip with the appropriate pH ranges positioned end on end 1n series see paragraph 3 2 az EG TO imensional j 3 1 Sample preparation Actually a unique 2 DE protocol of sample preparation that can be applied to all types of samples does not still exists In fact although several standard protocols have been published these methods have to be adapted and further optimized for the type of sample e g microbial mammalian cells to be analyzed as well as for the type of proteins of interest e g soluble or i
19. imensional 7 Recipes Table 1 IPG strip rehydration stock solution 8 M urea 2 CHAPS 0 5 2 Ampholyte Buffer 0 00290 bromophenol blue Store in aliguots at 20 C CHAPS 290 w v 0 5 g Ampholyte Buffer 0 5 v v 125 ul same range as the IPG strip stock solution Lu EW jegw Table 2 Thiourea rehydration stock solution 7 M urea 2 M thiourea 2 CHAPS 0 5 2 Pharmalyte or IPG Buffer 0 00290 bromophenol blue Store in aliguots at 20 C Thiourea FW 76 12 2M 3 8 g CHAPSi 2 w v 0 5 g same range as the IPG strip solution Powie ogw 7 DTT is added just prior to use 7 mg DTT per 2 5 ml aliguot of rehydration stock solution Other neutral or zwitterionic detergents e g Triton X 100 NP 40 may be used at concentrations up to 290 w v Note For the solubilization of more hydrophobic proteins it is recommended to use the urea thiourea buffer instead of the urea buffer 32 y SP re TF Dimensional 8 Technical specifications HIGH VOLTAGE POWER SUPPLY LOW VOLTAGE POWER SUPPLY for EWS 1 only Max voltage output Voltage resolution Current resolution 1 constant voltage Operative mode 2 constant current 3 constant power OTHER PARAMETERS Working temperature range 10 30 C Max humidity lt 90 not condensing 33 D 0 OFO R Tr Wa imensional 9 Ordering information
20. ks e After positioning of both the electrodes completely cover the IPG strips with the Cover Fluid e The instrument must be placed on a flat surface and the safety lid must be properly closed before power is applied Turning on the mains power switch located on the side of the EWS 1 and of the P Focus activates a self diagnostic program that runs for approximately 10 seconds CAUTION Always wear protective gloves when working with IPG strip Note After IEF IPG strip can be stored at 20 C or lower for several days For 2 DE applications SDS strip equilibration must be performed immediately prior to the second dimension run never prior to storage 13 FU ELETTROFOR TDi f 2 Scientific Instruments CN L m E n 5 L O nm a 4 Low voltage electrophoresis applications for EWS 1 only e The IEF tray can be removed and replaced with small electrophoretic chambers for other applications besides IEF such as the electrophoresis of DNA on agarose gels an example is reported in figure 8 e Connect the electrodes to the low voltage module Figure 8 14 WELEMTROEOR TU f Stcfen tific Instruments L m E n 5 L O n Qa 5 IEF Control Software manual Figure 9 DESCRIPTION A LCD Display G increase key selection ENTER B Control keyboard H decrease key selection ENTER C key LEFT lt I START key D key UP A J green LED if on machine is working E key DOWN V K key ST
21. located since the begin of a protocol PROTOCOL PAUSE AND STOP When a protocol is running you can pause the system by pressing the STOP PAUSE In this situation voltage on strip is cut off and the plate temperature is controlled When pause a protocol it displays the word PAUSE To exit the pause and resume the execution of the program press START button but 1f you press another time the STOP PAUSE the program end CURRENT CONTROL When a protocol is running an automated check is constantly made to the current The maximum current allowed through the strip is given by the product of the number of strip STRIP N and the value of current per strip CURRENT STRIP When system detects that the amount of current flowing through the strip is greater than the desired maximum current the applied voltage is decreased to the value that satisfies the constraint of current Current that crosses the Strip lt Strip Current Number of Strip See figure 11 for example The voltage limitation due to the current control is highlighted by the flashing of an asterisk written next to VOLT 20 ye ATE PETROFOR TT f Scenic a a au K Z f L m amp nm 4 L O nm a Max voltage value real limited value Current per Strip x Strip number desired value Figure 11 PROTOCOL EXECUTION END DATA STATISTICS e The end of protocol is indicated by a seguence of three beeps display will show RUN C
22. mpletely before use If wetted unplug the power supply until the instrument is dry e Use only undamaged electrical wire and equipment specified for the voltages you will use e High voltage electrical wires should be in accordance with IEC 1010 2 031 1993 All equipment connected to high voltage should be in accordance with IEC 1010 1 1993 y E a ELETTROFOR Tir f E A cen tific Instruments f L m E nm 4 L O n a 2 Function and description gt Electrophoretic WorkStation 1 The Electrophoretic WorkStation 1 EWS 1 is an innovative apparatus in the field of electrophoresis Millioni et al 2010 It is a versatile modular system which offers a solution for analytical or preparative separations of proteins and peptides by isoelectric focusing IEF on immobilized pH gradients IPG strip protein SDS PAGE DNA electrophoresis For IEF applications this instrument can accommodate 1 a shorter tray with twelve channels to perform IEF on up to 24 cm long IPG strips 11 a longer tray with six channels to perform IEF on up to 42 cm long IPG strips or shorter IPG strips of appropriate pH ranges positioned end on end The EWS 1 figure 1 includes the following features two integrated power supplies 1 High voltage power supply for IEF up to 15000V and 2mA The high voltage power supply is able to deliver a field strength of up to 15000 V a necessary feature for utilizing IPG strips longer than 25 30 cm In fact
23. nd pause After setting the desired parameters press START for a few seconds RUN will appear in the status bar REAL VALUES table will indicate actual values of voltage current and power lt is possible to PAUSE the protocol at any time by pressing STOP button To resume press START the power is turned on again and system will re start working If you used timer mode you can always stop manually the operations 28 F 7 ELETTROROR TF Os j a I IRSOHOMETFR imensiona 5 8 Error messages In case of faulty errors an audible and visual alarm will appear Error We code Contact your local distributor or the EEPROM error memory problem y manufacturer Temperature error temperature Contact your local distributor or the system problem manufacturer Error on the voltage value measured by high voltage module IEFSYSTEM Without any protocol running is measured a voltage different from zero Contact your local distributor or the manufacturer Q9 Error on the voltage value measured by high voltage module IEF SYSTEM During the execution of Contact your local distributor or the a program the voltage applied to manufacturer the strip is zero and cannot reach the set value Turn off the device wait some seconds and then turn on again Check the following parameters current strip voltage step number of strip set Try to run again the program If the error recurs contact
24. nsoluble membrane proteins However some general recommendations can be given e desalt the sample or prepare the sample so that the salt concentration is less than 10 mM Salt may be removed either by spin dialysis or by precipitation of proteins e g by TCA or organic solvents e samples containing urea must not be heated lt 37 C in order to avoid the carbamylation of the proteins by isocyanate formed in the decomposition of urea Isocyanate covalently modifies lysine residues thus inducing a change in isoelectric point e since IEF in the presence of 8M urea at low temperatures is not suitable due to the formation of urea crystals optimum focusing temperature is 20 C e proteases present within samples have to be inactivated by using protease inhibitors e delipidation can be obtained by extraction with organic solvents such as ethanol or acetone e unless present at low concentrations nucleic acids have to be removed by TCA acetone protein precipitation or by protease free RNAses and DNAses digestion e the most used sample solubilization buffer are the modified O Farrell lysis buffer 9 M urea 2 4 CHAPS 1 DTT 2 v v carrier ampholytes or the thiourea urea lysis buffer 2M thiourea 5 7M urea 2 4 v v CHAPS and or sulfobetaine detergents 1 DTT 2 v v carrier ampholytes e the minimum protein concentration should not be less than 0 1 mg ml and optimum concentration is 1 5 mg ml e long time storage of solubiliz
25. ny individual step length included in the protocol and the length of the rehydration phase if included PROGRAM NAME When cursor is moved inside the rectangle containing the name of the program it is possible to change individually any of the 16 letters that compose the name Pressing the and let you scroll in a loop the following character sets 22 a T Dimensional REHYDRATION PHASE A protocol may be preceded or not by the rehydration phase Move the cursor next to the written REHIDRATION and select YES to include it NO to exclude this phase STEP SET UP VOLT step voltage 10 15000 MIN 00 00 C TIME lt step lenght MAX 99 59 MODE operative mode r mee MUN See PROTOCOL END STEP below See JUMP OF A STEP below Note If a protocol is programmed with less than 10 STEP the cursor can be positioned on the first STEP zero volts but not to the next step see PROTOCOL END STEP For example if step 4 is the first null step is not possible to see next 6 10STEP STEP OPERATIVE MODE Each step can be programmed 1n 2 different mode e STEP AND HOLD During the execution of a protocol when a STEP programmed in this mode become active the voltage applied to the strip is brought guickly to the value set in the appropriate field and is maintained for the duration of the STEP e GRADIENT 23 D Ba D Dimensional During the execution of a protoc
26. ol when a STEP programmed is this mode become active the voltage applied to the strip is carried gradually from the initial VOLT value to the desired VOLT value set in the appropriate field The system is designed to work with increase and decrease operative mode figure 12 an illustrative example STEP 1 STEP 2 STEP 3 Figure 12 PROTOCOL END STEP If a VOLT value is set to zero all the parameters associated with the STEP and subsequent periods are replaced by symbol This STEP is the first null step of the protocol and indicates the end of it In figure 13 the last step of the protocol is STEP 3 JUMP OF A STEP If the value of TIME STEP is 00 00 it means that that STEP will be skipped during the protocol execution 24 l a IF Dimensional In Figure 13 at the end of step 1 STEP 3 will start STEP VOLT V TIME HH MM 1 ODE J Figure 13 5 6 3 Setup Within this section you can set parameters related to the rehydration phase and other general parameters The parameters modified in this section are written in memory and are used in all next work sessions A REHIDRATION PARAMETERS MIN 00 00 DURATION rehydration lenght hh mm MAX 99 59 TEMPERATURE rehydration temperature VOLTAGE rehydration voltage applied to the strip 25 7 ELETTROFEOR Td r f SV serre Instroments imenstona B GENERAL IEF PARAMETERS Parameter Measuring unit TEMPERATU
27. tected call Elettrofor service Press the switch on the right side of the device After turned on the system will check and test all the components for a few seconds The LCD display will show the company logo both LEDs will be lit see figure 9 J and L and an audible signal will be activate When test operations will be complete display will show MAIN MENU To turn off the system simply press the switch on the right 5 4 How to use the system The EWS 1 Electrophoresis WorkStation has two internal power supplies that can be used in 2 different exclusive mode 1 High voltage power supply for isoelectric focusing system IEF up to 15000V and 2mA 2 Low voltage power supply for normal electrophoresis up to 250V and 450mA 5 5 Main Menu Allowed selections in the MAIN MENU screen 1 IEF SYSTEM section relating to the high voltage 2 POWER SUPPLY low voltage power supply section Using UP and DOWN arrow keys move to the desired menu item To confirm press one of the selection key button 5 6 IEF system In this section you can access all functions for general setup protocol setup and the use of the high voltage power supply In the menu IEF MENU the following selections are allowed OPERATING STAGE PROGRAM SETUP SETUP Use the UP and DOWN arrow keys move to the desired menu item To confirm press one of the selection key button 17 AE EG TO imensional j 5 6 1 Operating Stage MAIN FEATURES e In
28. trip 3 6 11cm 1 5mm thickness 12 strips PRO 3 102412 IPG strip 3 10 24cm 1 5mm thickness 12 strips PRO 310NL2412 IPG strip 3 10 NL 24cm 1 5mm thickness 12 strips PRO 6102412 IPG strip 6 10 24cm 1 5mm thickness 12 strips PRO 582412 IPG strip 5 8 24cm 1 5mm thickness 12 strips PRO 362412 IPG strip 3 6 24cm 1 5mm thickness 12 strips 34 Z ELETTROFOR Stcren tric Jes FLYDD ts TD imenstonal PRO URE Urea MB grade 500g ci P mIDMIJDDJDMDMDMLZLoDoMDopD2 2 a Ampholyte Buffer SERVALYTTM 6 9 10ml SERVALYTTM 6 9 25ml SERVALYT 6 9 2ml SERVALYTTM 3 10 10ml SERVALYTTM 3 10 25ml SERVALYTTM 3 10 2ml SERVALYTTM 4 7 10ml PRO CHP25 CHPAS 25 gr PRO CHP100 CHPAS 100 gr PRO DTT DTT dithiothreitol MB grade 5g IAA iodoacetamide 5g PRO IAA25 IAA iodoacetamide 25 PRO TRX Triton X 100 MB grade 250ml Tris MB grade 1 Kg Molecular Weight Markers Proteome markers kit Svials PRO AGA Agarose for DNA electrophoresis 500g PRO GLE Glycerol MB grade 1L PRO BRB Bromophenol Blue 25g 35 q 27 amp ts Td Dimensional 10 References e Cargile BJ Bundy JL Freeman TW Stephenson JL Jr 2004 Gel based isoelectric focusing of peptides and the utility of isoelectric point in protein identification J Proteome Res 3 1 112 119 e Cargile BJ Sevinsky JR Essader AS Stephenson JL Jr Bundy JL 2005 Immobilized pH gradient isoelectri
29. ubilization buffer for peptide IEF is 8 M urea 1 DTT 5 v v glycerol 3 2 Rehydrating and loading IPG strips e IPG strips must be rehydrated prior to IEF Usually protein sample can be applied either by including it in the rehydration solution or by applying it directly to the rehydrated IPG strip via sample cups We suggest to include the sample in the rehydration solution because respect to the cup loading approach this method is technically simpler and eliminates the risk of precipitate formation which often occurs at the application point of cup loading The rehydration stock solution can be diluted no more than 1 8 by sample addition The amount of protein that can be added 1s dependent upon the length of strip the pH range and the detection method to be used e Select the eguilibration tray Tigure 2 corresponding to the IPG strip length chosen for the experiment Wash the extensively with double distilled water to Ep amp Ba D Dimensional remove residual protein The equilibration tray must be completely dry before use j n ne ME LT Le ns ji Figure 2 Equilibration strip tray for 7 cm long IPG strips Other different trays are available depending on the different length of IPG strips e Deliver the solution slowly at a central point in the rehydratation strip tray channel and remove air bubbles e Remove the protective cover from the IPG strip figure 3 F
30. with such a power supply it is possible to perform IEF on very long immobilized pH gradients at a voltage gradient of 330 V cm a value comparable to present day power packs operating at 8000 V on 24 cm long strips 2 Low voltage power supply up to 250V and 450mA A detailed description of the characteristics of these power supplies is reported in paragraph 8 temperature control by integrated Peltier elements the integrated control software with up to ten user defined IEF protocols each with up to ten steps per protocol Programmable functions include rehydration time 7 ELETTROFOR Tr f r r f SP nsttrements imensiona platform temperature current limit voltage limit for each step voltage gradient or step and step duration a set of strip tray for IPG strips rehydration running and equilibration These trays differ in length and number of strip channels The EWS 1 must be placed on a flat surface and the safety lid must be properly closed before power is applied Figure 1 Electrophoretic WorkStation 1 1 connectors of the high voltage module IEF system 2 connectors of the low voltage module 3 safety lid 4 control panel gt P Focus The only difference between the P Focus and the EWS 1 is the absence of the low voltage module 3 Isoelectric focusing applications Isoelectric focusing IEF is a technigue for separating molecules by their electric charge differences It
31. your local distributor or the manufacturer Current Error on the IEF SYSTEM module current The current that pass through the strip has exceeded the maximum output allowed by the device Error on low voltage module POWERSUPPLY Without any Contact your local distributor or the protocol running is measured a manufacturer voltage different from zero Error on the voltage value measured by low voltage module POWER SUPPLY During the execution of Contact your local distributor or the a program the voltage applied to manufacturer the strip is zero and cannot reach the set value Un gt 20 q BY amp i ZIK f s Dimensional Turn off the device wait some seconds and then turn on again Check if any short circuit are present or check if the load has a very low Current Error on the POWER SUPPLY module current The current absorbed has exceeded the maximum current allowed by the device impedance Try to reactivate the power supply If the error recurs contact your local distributor or the manufacturer 30 y y 17 ZAN TO imENSLO nal 6 Troubleshooting Probable reason Suggested solution The current is zero Incomplete contact between Make sure that the IPG strip the electrodes and the paper the electrodes and the paper bridge incomplete contact bridges are placed correctly between the paper bridge the IPG strip is not and the IPG strip the lid is
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