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IEF100 User Manual – English

1. Optional Add carrier ampholytes such as SERVALYTS to a concentration of 2 viv 20 ul per ml sample buffer solution e p53 e p54 IPG Strip Rehydration Solution 2A Urea Rehydration Stock Solution Prepares 50 ml 8 M urea 2 CHAPS 0 002 bromophenol blue Final Concentration Amount Urea FW 60 06 8M 24g CHAPS 2 wiv 1 08 Bromophenol blue 0 002 1 mg Deionized water to 50 ml Store in 3 ml aliquots at 20 C or below 3 ml is sufficient to rehydrate six 24 cm IPG strips Just prior to use for IPG strip rehydration e Add 0 5 2 0 v v carrier ampholytes SERVALYTS e Add 9 mg DTT per 3 ml aliquot of rehydration stock solution 20 mM DTT e Protein sample can also be added to the 3 ml of rehydration solution 2B Thiourea Rehydration Stock Solution Prepares 50 ml 7 M urea 2 M thiourea 2 CHAPS 0 002 bromophenol blue Final Concentration Amount Urea FW 60 06 7M 21g Thiourea FW 76 12 2M 7 6g CHAPS 2 wiv 1 0g Bromophenol blue 0 002 1 mg Deionized water to 50 ml Store in 3 ml aliquots at 20 C or below 3 ml is sufficient to rehydrate six 24 cm IPG strips Just prior to use for IPG strip rehydration e Add 0 5 2 0 v v carrier ampholytes SERVALYTS e Add 9 mg DTT per 3 ml aliquot of rehydration stock solution 20 mM DTT e Protein sample can also be added to the 3 ml of rehydration solution Note IPG strips should be equilibrated just prior to second dimensin PAGE Do
2. Check that the IPG strips have been fully hydrated prior to use Ensure the electrodes are connected to the corresponding receptacles Low starting voltage of gradient steps may not produce enough current to register at the beginning of the step Check the current in the strips again in a few minutes Run diagnostics to check the internal power supply Unit reads O pA O W for only one strip Poor IPG strip rehydration inspect the thickness height of the IPG strip over its entire length Check for arcing or burning that could break the electrical circuit Broken wire in the electrode Replace electrode No voltage output Run diagnostics from the options menu Contact your local Hoefer Inc representative Blank display Turn unit off and on Contact your local Hoefer Inc representative Screen frozen Turn unit off and on Contact your local Hoefer Inc representative Fails diagnostics The diagnostics program will check the volt and current output of the internal power supply the eprom and the system clock Contact your local Hoefer Inc representative if any of these tests come up negative e p43 Problem Solution Set voltage not reached Continue with the IEF IPG strip is being limited by pA or W Conductivity of the strip is too high One IPG strip will focus at its max pA or W and limit the voltage to all strips This stri
3. Run Screen The information on the run screen is described in detail in the IEF operation section The run screen has no editable fields It does have the following four soft keys MAIN PAUSE NEXT and GRAPH Fig 12 A Agioeter IEF100 ws Protocol 1 7cm broad amp basic running 6 strips Step 3 of 4 6000V Grad 8000 Vhrs Total 2 30 Hrs 5375 Vhrs Step 1 30 Hrs 4625 Vhrs Ela psed 4000V 300uA 1 2W Total 19 9 C Strip 50 uA 0 2W 50 uA 02W 1 2 3 50 UA 0 2W 4 50 UA 0 2W 5 50 uA 0 2W HIGH 9 VOLTAGE 6 50 UA 0 2W MAIN Allows the user to review and edit all protocols while the current TEF is still running PAUSE The PAUSE button interrupts the high voltage to the IPG strips allowing the user to interface safely with the strips After a pause RUN will continue from where it was interrupted or ABORT will terminate the run NEXT The NEXT button allows the user to skip the remaining portion of the active step and start the next step in the protocol GRAPH The GRAPH button will display the current run results graphically Use the knob to scroll through the voltage profile and the pA profiles in channels 1 6 Press EXIT to return to the run screen At the end of the run the IEF100 will beep and turn off the high voltage power supply A screen displays the protocol number the end date and time and the final TEF conditions in each IPG strip Note When the optio
4. 0 30 Hrs Step 2 Gradient volt 1000 V 0 30 Hrs Step 3 Gradient volt 6000 V 0 30 Hrs Step 4 Constant volt 6000 V 8000 Vhrs Step 5 Constant volt 1000 V 1 00 Hrs Program 2 Focusing of 7 cm IPG S under constant watt conditions Name 7 cm const watt Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 6000 V 0 5 W Step 1 Constant watt 0 1 W 1 00 Hrs Step 2 Constant watt 0 5 W 8000 Vhrs Step 3 Constant volt 1000 V 1 00 Hrs Program 3 Focusing of broad and medium 18 cm IPG s by limiting volt parameters Name 18 cm broad amp basic Delay 0 00 Delay temp 20 C Run temp 20 C 500uA 12000V 1 5W Step 1 Gradient volt 1000V 1 00 Hrs Step 2 Gradient volt 12000V 1 00 Hrs Step 3 Constant volt 12000V 25000Vhrs Step 4 Constant volt 1000V 1 00 Hrs Program 4 Focusing of 18 cm narrow pH range IPG s by limiting volt parameters Name 18cm narrow Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 12000 V 1 5 W Step 1 Gradient volt 1000 V 1 00 Hrs Step 2 Gradient volt 12000 V 1 00 Hrs Step 3 Constant volt 12000 V 50000 Vhrs Step 4 Constant volt 1000 V 1 00 Hrs Program 5 Focusing of 18 cm IPG S under constant watt conditions Name 18 cm const watt Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 12000 V 1 5 W Step 1 Constant watt 0 1 W 1 00 Hhrs Step 2 Constant watt 1 5 W 25000 Vhrs Step 3 Constant volt 1000 V 1 00 Hrs Program 6 Focusing of broad and medium r
5. Horizontal Arrow Keys High Voltage LED STOP Button LCD Display Function Keys Ethernet Port RS232 port e pd O STOP Button Immediately stops the IEF and terminates the protocol LCD Display The large blue white display simplifies the interface and displays graphical IEF results Function Keys The function of these keys is shown on the display directly above the key The function varies depending on what screen is active Ethernet Port RS232 Port These two ports can be used to download IEF data from the instrument and transfer protocols Air Flow Fans pull air in through the side vents and blow air out the rear vents The air cools the electronic components and helps the Peltier modules maintain constant temperature Mains Power The power entry module operates at all volts and frequencies Leveling Feet Bubble Level The leveling feet and the bubble level can be used to level the IEF platform A level instrument will help ensure oil completely covers the IPG strips Fig 5 Rear panel Leveling Feet Note The vents should not be blocked Air Out Vents Air In Vents Power Entry 2X Module ON OFF Switch p6 Fig 6 Top of the instrument Focusing Tray The 6 channel focusing tray accommodates IPG strips up to 24 cm in length Small grooves in the tray allow for easier removal of the IPG strip after focusing Detail of these features can be seen on page 24 The channels
6. Oververhittend zal onherstelbare schade aan de eenheid veroorzaken T rke Tietoa Finnish Jos t t varusteita k ytet n tavassa ei m ritetty Hoefer Inc suojelu ehk isty varusteille saattaa olla avuton T m v line suunnitellaan sis laboratoriok yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoefer Inc oheen voi k ytt k ytt miselle valvoalle ja servicing t m tuote e Vain k ytt k ytt j nnitett joka on CE merkitsi tai turvallisuus joka on todistanut aidoksi ohi joka on kansallisesti tunnustettnut testaaminen laboratoriota Turvallisuuskansi t ytyy olla paikallaan ennen yhdist minen e pii kaytt j nnitelyijyj k ytt j nnitteeseen Kiert kaikki k ytt j nnitevalvonnat ja irrottaa valtalyijyt ennen poistaminen turvallisuuskantta Kiert vain vesi tai 50 50 vesi ethylene glycol siin tapauksessa varustetun l mm nvaihtimen l pi l yhdist limm nvaihdinta vesinapautukseen eik j hdytysnestel hteeseen miss vesipaine on unregulated Pakkasneste eik orgaaninen liuotin v lineen osassa ei esitele Koskaan Orgaaniset liuottimet aiheuttavat korvaamattoman vahingon yksikk n Ei k yt puskuria yll olevia l mp tiloja enint n m ritetyill teknisill t smennyksill Ylikuumeneminen aiheuttaa korvaamattoman vahingon yksikk n Information Importante French Si cet quipement est utilis dans une
7. 0 00 narrow 6 S 0 H 0 00 narrow 7 S 0 H 0 00 narrow 8 8 0 H 0 00 narrow 9 S 0 H 0 00 IPROT 8 24cm const Watt D 0 00 20 20 500 12000 2 0 IPROT 8 24cm const Watt 1 W 0 1 H 1 00 IPROT 8 24cm const Watt 2 W 2 0 V 45000 IPROT 8 24cm const Watt 3 S 1000 H 1 00 IPROT 8 24cm const Watt 4 S 0 H 0 00 IPROT 8 24cm const Watt 5 S 0 H 0 00 IPROT 8 24cm const Watt 6 S 0 H 0 00 IPROT 8 24cm const Watt 7 S 0 H 0 00 IPROT 8 24cm const Watt 8 S 0 H 0 00 IPROT 8 24cm const Watt 9 S 0 H 0 00 Protocol 9 IPROT 9 24cm cup load D 0 00 20 20 500 12000 2 0 IPROT 9 24cm cup load 1 G 1000 H 4 00 IPROT 9 24cm cup load 2 G 12000 H 6 00 IPROT 9 24cm cup load 3 W 2 0 V 64000 IPROT 9 24cm cup load 4 S 1000 H 10 00 IPROT 9 24cm cup load 5 S 0 H 0 00 IPROT 9 24cm cup load 6 S 0 H 0 00 IPROT 9 24cm cup load 7 S 0 H 0 00 IPROT 9 24cm cup load 8 S 0 H 0 00 IPROT 9 24cm cup load 9 S 0 H 0 00 Common classes of additives Denaturants Unfold the proteins to expose the internal native charges Non lonic Detergents Make samples more soluble without altering the protein charge Reductants Help break internal disulfide bonds to further unfold the proteins and help reduce the negative effects of oxidation of proteins during rehydration and IEF Other Carrier amphoytes proteases DNases RNases Appendix B Reagents and Solutions Sample Preparation Samples prepared for 2D should be fully denatured free of insoluble material and low in ove
8. Fig 8 Sample cups Fig 9 Rehydration equilibration trays Electrode Wicks The electrode wicks are small rectangular filter papers that are placed over the end of the IPG strips The electrodes make contact with the top of the wicks The electrode wicks help remove impurities that collect at the end of the IPG strips and generally improve electrode contact The electrode wicks come in long strips and must be cut apart into individual rectangles as they are used The electrode wicks need to be moistened by lightly blotting with water prior to use Sample Cups The sample cups load protein into the IPG strip Sample cups can hold up to 240 pl of sample The cups can be applied in most places along the length of the IPG strip and are normally applied at the anode end Rehydration Equilibration Trays The rehydration equilibration trays can be used for both functions The lanes are narrow enough for proper rehydration but deep enough to hold the volume of reagent required for equilibration A small tray is included for use with 7 cm IPG strips The large tray can be used with IPG strips up to 24 cm in length Brush The cleaning brush is used for cleaning the focusing tray Forceps Forceps facilitate handling of IPG strips e p9 Fig 10 Main screen e p10 Programming the IEF100 Main Screen When turned ON the main screen is displayed Fig 10 The main screen contains the following fields D
9. The volt and current graphs can not be printed directly from the IEF100 If data output is sent to a computer the data can be transferred to a program such as Excel to create the graph The Four Buttons EDIT RUN OPTION and GRAPH EDIT Edits the highlighted protocol RUN Starts the highlighted protocol OPTION Allows access to port settings and the date and time GRAPH Will display the volt and measured currents graphically Use the knob to scroll through the voltage and the pA profiles in channels 1 6 The active run will be displayed If not actively running a protocol the graph button will display the last run data e p11 Fig 11 Edit protocol screen All parameters for an entire protocol are viewable on one screen e p12 Edit Protocol Screen From the main menu use the knob to highlight the desired protocol Press the button beneath EDIT on the screen The IEF100 will now display the edit protocol screen Fig 11 All IEF parameters are displayed on one screen Each protocol can be programmed with up to nine steps The protocol ends with the first step that has a step time of zero A uoeter IEF100 nor Protocol 1 7cm broad amp basic Delay 0 00 Hrs 20 C IEF Limits 6000V 500uA 0 5W str 20 C Ste 1 Gradient Volts 500V 0 30 Hrs 2 Gradient Volts 1000V 0 30 Hrs 3 Gradient Volts 6000V 0 30 Hrs 4 Constant Volts 6000V 8000 Vhrs 5 Constant Volts 1000V 1 00 Hrs 6 Constant Volts 7 C
10. connection p20 IEF100 Operation Using the Knob The knob has two actions it turns and pushes in to click Fig 14 Turn Navigates through fields adjusts values Click Changes between MOVE and SET modes as indicated by the LED 1 In MOVE mode highlight the field that you want to change 2 Click the knob to change to SET mode 3 Turning the knob will now adjust the value of the field When the proper value is shown click the knob again to enter the value and change back to MOVE mode Turning the knob will now move to the next field IEF100 Setup 1 Plug the IEF100 into a grounded mains outlet using the appropriate power cord An adapter may be necessary in some countries The power entry module is on the rear panel The ON OFF switch is located on the power entry module Fig 15 2 Allow for clearance around instrument and enough clearance above to allow for opening the lid The vents should not be blocked Do not allow fluids to enter the side intakes 3 Wash the electrode tray rinse well with deionized water and allow to air dry 4 Gently clean electrode platinum wire contact points and allow to air dry 5 When the unit is inactive for a period of time a screensaver will be displayed to prolong the life of the display Push any button to exit the screensaver Printer and Computer Connections Attach and configure a printer or a computer before focusing and the IEF100 automatically sends output data in
11. in the tray are numbered 1 6 from front to back of the IEF100 The tray fits in the instrument in only one orientation It is set in place on the right side of the platform and moved sidewise to the left clamping the tray down against the cold plate The clamping mechanism improves the heat transfer for the plastic trays There are and marks on the tray and the instrument to help with proper orientation Electrode gt Electrode Sample Cups Focusing Tray Clamps Align IEF Strips with this Line Clamping Tabs 6X Front of Instrument e p7 Fig 7 Positive electrode top and negative electrode bottom e p8 Electrodes Positive Electrode Anode A high volt connector attaches the positive electrode s to the high volt terminal The anode has a single white wire and connector and locates on the left side of the tray Negative Electrode Cathode A six wire lan connector attaches the negative electrode s to the ground terminal monitoring the current in each lane The cathode locates on the right side of the tray Electrodes should be placed in the tray with the number 1 towards the front of the tray and the platinum wire electrodes facing the middle of the IPG strips Note The electrodes will not make contact with the IPG strips without the electrode wicks in place Note The sample cups can be cut into individual cups if desired
12. la protezione fornito dall apparecchiatura potrebbe essere indebolita Questo strumento disegnato per l uso di laboratorio interno solo Solo gli accessori e le parti hanno approvato o hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare questo prodotto usa Solo un alimentatore che CE ha marcato o la sicurezza certificato da un nazionalmente riconosciuto testando il laboratorio Il coperchio di sicurezza deve essere nel luogo prima di collegare i piombi di alimentatore a un alimentatore Spegne tutto i controlli di alimentatore e disinserisce i piombi di potere prima di togliere il coperchio di sicurezza Circola solo l acqua o 50 50 glicole di acqua etilene attraverso lo scambiatore di calore se cosi equipaggiato Non collegare lo scambiatore di calore a un rubinetto di acqua o qualunque fonte di refrigerante dove la pressione di acqua sregolata Non introduce mai l antigelo o qualunque solvente organico in qualunque parte dello strumento solventi organici causeranno il danno irreparabile all unita Non opera con le temperature di tampone al di sopra del massimo ha specificato le descrizioni tecniche Il surriscaldamento causer il danno irreparabile all unita Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesifisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innen
13. place Place the electrode on top of the electrode wicks so that the platinum wire is centered in the area of overlap between the wicks and the IPG strip Fig 24 The electrode should lightly lock into place Fig 23 Connect electrode Fig 24 Lock electrode in place e p25 Fig 25 Connect electrode Fig 27 Cover with mineral oil e p26 O Connect the electrode to the terminal Fig 25 Place the electrode on top of the electrode wicks so that the platinum wire is centered in the area of overlap between the wicks and the IPG strip Fig 26 The electrode should lightly lock into place O Cover the strips and any empty channels with 60 ml mineral oil Using less oil will risk that IPG strips will not be completely covered and may dry out during the run Fig 27 Fig 28 IEF100 set up using the Dual Electrode Accessory Isoelectric Focusing using the Dual Electrode Accessory The Dual Electrode Accessory increases the Hoefer IEF100 capacity enabling up to twelve 7 cm IPG strips to be run simultaneously This is accomplished by running up to six pairs IPG strips in parallel Each of the six channels will report the sum of the current and wattage of the paired strips therefore the current and wattage limits should be set at twice the usual value used when running single strips and as always the IPG strips being run simultaneously must be of the s
14. protein sample to IPG strips if it was not included in the rehydration buffer The sample cups are supplied in strips of six cups The sample cups can be used all together or cut apart and used separately Press the sample cups down on top of the IPG strips The sample cups should stop on the bottom of the channel and seal on the IPG strips Fig 30 In general most cup loading is done on the anodic end of the IPG strip Each sample is different In some situations samples may focus better loaded elsewhere on a strip This can only be determined experimentally for different sample types o Up to 240 ul sample can be applied to the sample cups Fig 31 e Once loaded confirm the protocol settings o Close the lid Fig 32 e p29 Note The protocol s overall limits of current strip and watt strip will not operate correctly without entering the proper number of channels containing strips Note The IEF100 monitors the current and watt in each channel If necessary conditions can be adjusted and or problematic strips can be removed from the run e p30 Start IEF 0 Highlight the desired protocol and press RUN o Confirm the number of focusing tray channels containing IPG strips Use the knob to change the value e Press RUN again to start the IEF 4 The IEF100 will beep to indicate the start of a run The high voltage LED will light and RUNNING will be flashing
15. tego produktu korzysta jedynie zasilacza e jest nosz ce oznakowanie CE lub bezpiecze stwa uwierzytelnione przez uznane na poziomie krajowym laboratorium badawcze Bezpiecze stwo lid musi by w miejsce przed pod czeniem zasilania prowadzi do zasilania Za wszystkie r d a zasilania urz dzenia steruj ce off i od czy moc prowadzi przed odbiorem bezpiecze stwa lid Kr tylko wody lub wody 50 50 ethylene glycol wymiennik ciep a poprzez je li tak wyposa one Nie nale y po czy wymiennik ciep a woda z kranu lub jakimkolwiek ch odziwo r d a je eli ci nienie wody jest nieuregulowanych Nigdy nie wprowadza rozpuszczalnika organicznego przeciw zamarzaniu lub jakichkolwiek na dowoln cz dokumentu Rozpuszczalniki organiczne spowoduje nieodwracalne szkody dla jednostki Nie dzia aj w buforze temperatury powy ej maksymalnego okre lone specyfikacje techniczne Przegrzania spowoduje nieodwracalne szkody dla jednostki Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protec o fornecida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S acess rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto S usa um estoque de poder que CE marcou ou seguran a registrada por um nacionalmen
16. temperature can be entered Focusing will start after the delay time has elapsed This can be helpful for overnight runs where the user may want to have focusing end at some specific time the next morning The Peltier modules will maintain the cold plate at the set temperature Parameter Units Range Increment Time hour min 0 00 99 59 0 15 Temperature C 15 25 1 IEF Limits Maximum volt current per strip and watt per strip are applied to the protocol These settings will override the values in the individual steps These overall limits can be used to avoid certain focusing conditions that cause damage or problems with the IPG strips The temperature for the focusing is also set Since the isoelectric points of proteins are temperature dependent IEF is typically at a single temperature usually 20 C It is not recommended to use temperatures lower than 20 C as it can cause problems with urea crystallization in the IPG strip Knob Computer Settable Settable Parameter Units Range Increments Increments Volt V O 12 000 250 1 Current strip uA O 999 25 1 Watts strip W 0 2 0 0 1 0 1 Temperature C 15 25 1 1 e p13 Note It is not recommended to set a series of discrete volt steps The discontinuities between steps cause spikes in power levels that tend to increase the chances of burning strips Note In some cases the volt settings may never be reached Note If a constant watt step is followed by a gr
17. 00 for focusing e p21 Note The IEF100 is a convenient place to rehydrate strips The cold plate will maintain a constant temperature and the cover will help protect the IPG strips from dust Note Using more than the recommended volumes may contribute to loss of protein or blurry IEF results Note Rehydration solutions typically contain high concentrations of urea that tend to crystallize at lower temperatures For this reason rehydration should not be done in cold rooms or in environments significantly colder than 20 C Note Sliding the strips in the rehydration buffer will wet the entire length of the strip and helps prevent sticking Strips placed directly against the plastic bottom of the rehydration tray may stick and not rehydrate properly e p22 Rehydration of IPG Strips A brief discussion of rehydration ingredients and their function is included in Appendix B along with some recommended solutions The reagents can be customized for specific samples The volume of solution is dependent on the length of the strip to be used Hoefer recommends following the IPG strip manufacturer s recommendations A table of typical volumes is included below Strip Length cm Volume per Strip ul 7 130 18 340 24 450 Apply rehydration solution in a channel of the appropriately sized rehydration tray in a line slightly shorter in length than the strip to be rehydrated Many IPG strip manufacturer s cover the
18. 1 7cm broad amp basic D 0 00 20 20 500 6000 0 5 IPROT 1 7cm broad amp basic 1 G 500 H 0 30 IPROT 1 7cm broad amp basic 2 G 1000 H 0 30 IPROT 1 7cm broad amp basic 3 G 6000 H 0 30 IPROT 1 7cm broad amp basic 4 S 6000 V 8000 IPROT 1 7cm broad amp basic 5 S 1000 H 1 00 IPROT 1 7cm broad amp basic 6 S 0 H 0 00 IPROT 1 7cm broad amp basic 7 S 0 H 0 00 IPROT 1 7cm broad amp basic 8 S 0 H 0 00 IPROT 1 7cm broad amp basic 9 S 0 H 0 00 Protocol 2 IPROT 2 7cm const Watt D 0 00 20 20 500 6000 0 5 IPROT 2 7cm const Watt 1 W 0 1 H 1 00 IPROT 2 7cm const Watt 2 W 0 5 V 8000 IPROT 2 7cm const Watt 3 S 1000 H 1 00 IPROT 2 7cm const Watt 4 S 0 H 0 00 IPROT 2 7cm const Watt 5 S 0 H 0 00 IPROT 2 7cm const Watt 6 S 0 H 0 00 IPROT 2 7cm const Watt 7 S 0 H 0 00 IPROT 2 7cm const Watt 8 S 0 H 0 00 IPROT 2 7cm const Watt 9 S 0 H 0 00 Protocol 3 IPROT 3 18cm broad amp basic D 0 00 20 20 500 12000 1 5 IPROT 3 18cm broad amp basic 1 G 1000 H 1 00 IPROT 3 18cm broad amp basic 2 G 12000 H 1 00 IPROT 3 18cm broad amp basic 3 S 12000 V 25000 IPROT 3 18cm broad amp basic 4 S 1000 H 1 00 IPROT 3 18cm broad amp basic 5 S 0 H 0 00 IPROT 3 18cm broad amp basic 6 S 0 H 0 00 IPROT 3 18cm broad basic 7 S 0 H 0 00 IPROT 3 18cm broad amp basic 8 S 0 H 0 00 IPROT 3 18cm broad amp basic 9 S 0 H 0 00 Protocol 4 IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm IPROT 4 18cm Prot
19. ANSI v Terminal Setup Colors Telnet terminal ID ANSI Backscroll buffer lines 500 Play sound when connecting or disconnecting Exit program upon disconnecting ASCII Setup 5 In the ASCII setup section check the boxes Send line ends with line feeds Echo typed characters locally Fig 43 6 Set the line delay to 30 milliseconds Set the character delay to O milliseconds 8 In the ASCII receiving section check the box Wrap lines that exceed terminal width Fig 43 ASCII Setup m ASCII Sending Iv Send line ends with line feeds Iv Echo typed characters locally Line delay 30 milliseconds Character delay fo milliseconds ASCII Receiving Append line feeds to incoming line ends Force incoming data to 7 bit ASCII Iw Wrap lines that exceed terminal width Cancel e p38 Note The data from the IEF100 is a series of text fields separated by commas One method to transfer data to other programs is to use the Windows clipboard to copy and paste the data yperTerminal MEMES Help Send File Receive File Capture Text Send Text File mem fF In Capture to Printer Fig 44 Capture Text Folder C allen Hyperterm File C Allen Hyperterm RUN1 1 27 TXT Browse Send File Receive File E ETC sv A Send Text File Pause Capture to Printer Note Copying and pasting the data will by default put all the data in
20. Mineral oil e p3 Fig 3 Instrument components e p4 Electrode Wicks Accessory Box IEF106 pa Level Forceps Sample Cups 1EF108 Electrode Brush Large Rehydration Equilibration Tray IEF110 Small Rehydration Equilibration Tray IEF109 4 Electrode Not pictured Dual Electrode Accessory IEF212 Focus Tray IEF105 230 V Power Cords EU and UK Note Turn the knob slowly to get desired movement on the screen Turning the knob too fast causes the cursor to blink and stay in place If this happens turn the knob slower until the proper behavior is observed Note The click action of the knob is to quickly push and release like a mouse click Do not hold the knob in Fig 4 Front of the instrument Component Descriptions Safety Lid The transparent safety lid must be closed in order for voltage to be applied to the electrodes Interlocks are used to stop the voltage output should the lid be opened during electrophoresis Knob The knob moves the cursor or changes the values of a given field Pressing the knob inwards to click switches between these two functions A LED indicates which function is active MOVE or SET Horizontal Arrow Keys The horizontal arrows are used when setting protocol names and the date and time fields High Volt Indicator LED This LED is lit when voltage is being applied to the electrodes Safety lid Knob
21. P Connection Using the Ethernet Port Use the IEF100 ethernet port to connect to a local area network LAN Plug a live ethernet cable into the port The LAN will automatically assign an internet address to the IEF100 The internet address is shown on the IEF100 options screen Ethernet Port Configuration 1 From the HyperTerminal properties screen select the TCP IP Winsock option at the communication port properties windows Fig 40 2 Press OPTION from the main screen of the IEF100 The local server should automatically assign an internet address in the XXX XXX XXX XXX format Enter the IP address to the IEF100 in the host address line in XXX XXX XXX XXX format Fig 41 3 Set the port number to 10001 Fig 41 TestCOM1 Properties HE HoeferTCP Properties EE Connect To Settings Connect To Settings Q recom eeu Country region Host address E 6 175 108 Enter the area code without the long distance prefix Area code Port number 10001 Phone number Connect using Connect using TCPAP Winsock Fig 40 Select the TCP IP option Fig 41 Enter IP address and port number e p37 EEFTYWRWPBRES a 4 Click on the SETTINGS tab and the ASCII Setup button Fig 42 Fig 42 TestCOM1 Properties HEI ConnectTo Settings Function arrow and ctrl keys act as Ter s Windows keys Backspace key sends Dk Del Ctl H Space Ctri H Emulation
22. Qus Hoefer IEF100 Isoelectric Focusing Unit OT A Ho efer Page Finder Important Informaticas roedor ii Waste Electrical And Electronic Equipment WEEE vi Function and Description ccoocccconcccncnncncnncnnonononnnnnnnncnnnnoninnnonos 1 Specification S ceteri tr e E CR EA 2 System COMPONENTS ege iude TE oues sas a EL 3 Component Descrtptions eee mene 5 Programming the VEF 100 eie rne nre aaa 10 IEF LOO Operation E 20 Optional Data Connections occccoccnccccncnonnnnncnonnnnnnncnnnnncncnncnonnns 35 Care and Maintenance sassaresi oira 42 Technical Service and Repair 42 Trouble Nica ia di anaie 43 Ordering Information aia ciao 45 Appendix A Preprogrammed Protocols ooocccocccccocccconncnnnncnonons 46 Appendix B Reagents and Solutions 51 Appendix C References IEF IO 57 pi Important Information English If this equipment is used in a manner not specified by Hoefer Inc the protection provided by the equipment may be impaired This instrument is designed for indoor laboratory use only Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Only use a power supply that is CE marked or safety certified by a nationally recognized testing laboratory The safety lid must be in place before connecting the power supply leads to a power supply Turn all power supply controls off and disconn
23. The protocol number and name The word finished is displayed in the upper right corner Line 2 The number of strips run or number of channels if using the Dual Electrode Accessory Line 3 The date and time that focusing terminated Line 4 The total time and volt hours of focusing Line 5 The last step time and volt hours Line 6 The volts and the total current and watts of all IPG strips at the end of the run Line 8 13 The conditions in each strip at the end of the run The results can be viewed graphically by pushing the function GRAPH The data will be retained in memory until a new run is started At this point the IPG strips can be stored at 20 C Or if the second dimension is to proceed immediately the buffer needs to be exchanged in a two step equilibration process before second dimension PAGE Connection Description HE AP New Connection Enter a name and choose an icon for the connection Name Icon Bosse DK Cancel Fig 37 Assign name to the new connection Optional Data Connections Serial Printer Connection Use the RS232 port to connect directly to a serial printer The printer and cable are not supplied The serial printer should have the following settings Baud Rate 9600 38400 or 57600 must match IEF100 baud rate setting Data Bits 8 Parity None Stop Bit 1 Flow Control None The following section describes the serial computer connection or a ethernet
24. adient volt step the gradient volt step will start from O and increase linearly to the set voltage e p14 Steps To edit any step parameter turn the knob to highlight the parameter Press the knob and turn the knob to set the desired value Press the knob again to enter the value and move to the next field Each step has four fields that can be edited the step type a step value a step time and step units Step Type Constant volt gradient volt constant watt Step Value Sets the maximum value of volt or watt depending on step type Step Time Time in hours or volt hours Step Units Hours Hrs or volt hours Vhrs Step Type and Step Value Each step can be programmed in one of three different modes constant volt gradient volt or constant watt e In constant volt the voltage remains constant thoughout the length of the step e In gradient volt the voltage will start at the end voltage of the previous step and increase or decrease linearly over time to the voltage entered in the current step If the first step of the protocol is a gradient volt step the voltage will increase from 0 Gradient volt steps gradually increase the power being applied to the IPG strips resulting in more even heating of the strip over time Constant watt steps will smooth out the heat generation as much as possible over the course of the IEF separation There will still be localized heating due to different ion concentrations alon
25. ame pH and conductivity Open the safety lid by pressing down on the push label in the middle of the front of the lid o Insert the IEF105 focusing tray on the far right side of the cold plate The tray has only one orientation There are and marks on the tray that align with the corresponding marks on the IEF100 The tray will not fit properly in the IEF100 in any other orientation e Slide the focusing tray to the left under the clamping tabs These tabs improve the contact and the heat transfer between the tray and the cold plate o Use forceps to load the rehydrated 7 cm IPG strips into the focusing tray with the gel side of the strips facing up The first set of IPG strips should be loaded on the left side of the tray with the anodic end of the strips on the left matching the on the tray and the IEF100 e p27 Fig 29 IPG Strip Placement e p28 O The second set of IPG strips are loaded to the right of the first set in the opposite orientation with the anodic end of the strips matching the on the tray and the IEF100 The cathodic end of the each set of strips should be facing each other in the center of the focusing tray approximately 4 cm apart Fig 29 9 Parallel IPG strips should be aligned as closely as possible using the or imprinted on the strip to guide alignment O Apply electrode wicks on top of each end of the IPG strips overl
26. ange 24 cm IPG s by limiting volt parameters Name 24 cm broad amp basic Delay 0 00 Delay temp 20 C Run temp 20 C 500 pA 12000 V 2 0W Step 1 Gradient volt 1000 V 1 00 Hrs Step 2 Gradient volt 12000 V 1 00 Hrs Step 3 Constant volt 12000 V 45000 Vhrs Step 4 Constant volt 1000 V 1 00 Hrs Program 7 Focusing of 24 cm narrow pH range IPG s by limiting volt parameters Name 24 cm narrow Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 12000 V 2 0 W Step 1 Gradient volt 1000 V 1 00 Hrs Step 2 Gradient volt 12000 V 1 00 Hrs Step 3 Constant volt 12000 V 100000 Vhrs Step 4 Constant volt 1000 V 1 00 Hrs Program 8 Focusing of 24 cm IPG S under constant watt conditions Name 24 cm const watt Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 12000 V 2 0 W Step 1 Constant watt O 1 W 1 00 Hrs Step 2 Constant watt 2 0 W 45000 Vhrs Step 3 Constant volt 1000 V 1 00 Hrs Program 9 Slow focusing of 24 cm IPG S with sample loaded into a cup Gently increases volt over time Sample entry phase is prolonged and designed to be an overnight run Name 24 cm cup load Delay 0 00 Delay temp 20 C Run temp 20 C 500 uA 12000 V 2 0 W Step 1 Gradient volt 1000 V 4 00 Hrs Step 2 Gradient volt 12000 V 6 00 Hrs Step 3 Constant watt 2 0 W 64000 Vhrs Step 4 Constant volt 1000 V 10 00 Hrs e p47 e p48 Pre programmed Protocols in Machine Code Format Protocol 1 IPROT
27. apping the gel by 2 3 mm and extending off the end of the IPG strip e The electrode wicks are supplied in long perforated strips Use scissors to cut off the desired number of wicks e Moisten the IEF wicks with water and gently blot off any excess water O Connect the anode electrodes to the positive terminal on the left side of the IEF100 The anode electrodes are placed on either end of the focusing tray the left side of the first set of IPG strips and the right side of the second set of IPG strips The electrodes should be centered on top of the area where the wick overlaps the gel of the IPG strip O Connect the cathode electrodes to the negative terminal on the right side of the IEF100 The cathode electrodes are placed in the center of the focusing tray on the right side of the first set of IPG strips and the left side of the second set of IPG strips The electrodes should be centered on top of the area where the wick overlaps the gel of the IPG strip on tray on tray IPG Strip IPG Strip A nho AY do CC Note There are small semicircular features in the bottom of the tray channels that help align the IPG strip in the middle of the lane The legs of the sample cup must not hit these features or else the sample cup will leak See Fig 21 on page 24 Fig 30 Place sample cups Fig 32 Close safety lid Sample Cups Sample cups can be used to apply
28. ate and Time The date and time are set up at Pacific Standard Time Use the OPTION button to set the date and time for your location See options screen page 17 List of 30 Protocols The first nine protocols are shown on the display One of the proto cols is always highlighted Use the knob to scroll through the other protocols The instrument is preloaded with 9 protocols for focusing 7 18 or 24 cm IPG s These are general guidelines for focusing IPG strip manufacturers normally give recommended focusing times in total volt hours volts multiplied by hours Both under and over focusing can be problematic Optimum focusing times should be determined by the end user considering the IPG strip the sample type and sample protein load All protocols can be overwritten if desired In addition to these 9 protocols there are 21 additional unnamed protocols without preprogrammed steps allowing the user ample space to create custom protocols A Apioeter 1EF100 no Hoefer IEF100 dd mmiyyyy hhimm Protocols 1 Zem broad amp basic 2 7cm const Watt 3 18cm broad amp basic 4 18cm narrow 5 18cm const Watt 6 24cm broad amp basic 7 24cm narrow 8 24cm const Watt 9 24cm cup load HIGH O VOLTAGE Y EDIT RUN OPTION GRAPH B Keesen Note The last run data is retained in memory until a new run is started Once a new run is started the last run data is no longer available Note
29. c end of the strips should be on the left matching the on the tray and the IEF100 Fig 19 The strip channels are numbered so that channel 1 is closest to the front and channel 6 is at the back of the IEF100 e p23 Fig 20 Align strips Note The electrode wicks absorb ions collecting at the ends of the IPG strips and generally improve the IEF results Fig 21 Focusing tray enlarged Fig 22 Wick placement e p24 Align the end of the strips with the alignment mark in the tray Fig 20 This will help in two ways First the clamping tabs will not interfere with the positioning of the electrodes Secondly there are small grooves in the bottom of the channels for forceps to remove IPG strips after IEF O Apply electrode wicks on top of each end of the IPG strips overlapping the gel by 2 3 mm and extending off the end of the IPG strip e The electrode wicks are supplied in long perforated strips Use scissors to cut off the desired number of wicks e Moisten the IEF wicks with water and gently blot off any excess water Grooves for Forceps to Remove IPG Strips IPG strip alignment features Do not use sample cup over IPG Strip Clamping these features Alignment Line Tabs Platinum Wire Electrode Wick O Connect the electrode to the terminal Fig 23 Note The electrodes will not make contact with the IPG strips without the O electrode wick in
30. can be added to aid in protein solubility and help prevent protein precipitation during focusing Concentrations of 0 596 2 v v are typically used Carrier ampho lytes may interfere with certain labeling experiments In those cases the reagents should then be omitted from the sample extraction step e p51 Note Salts are perhaps the most common contaminant causing poor IEF results e p52 General Guidelines Sample preparation procedures are being refined and standardized It is best to consult the literature to determine if a particular sample preparation buffer is recommended for the sample type EF works best with pure protein samples that are solubilized and denatured and free of interfering molecules Remove insoluble material with centrifugation Keep the salt content as low as possible Use freshly prepared reagents of high quality or reagent solutions that have been stored frozen Do not leave urea solutions out at room temperature for extended periods of time Never heat protein samples in urea solutions Heating cause carba mylation of proteins and will alter the native charge of the proteins Keep samples on ice to prevent degradation Add protease inhibitors to prevent protease activity Protease inhibitors such as PMSF or Pefabloc can be added to inhibit serine protease activity while pepstatin can inhibit aspartic proteases Some protocols use basic carrier ampholytes or Tris to
31. connection to the IEF100 HyperTerminal Connection HyperTerminal is a Windows program that can communicate with external devices By setting up a connection between a computer and the IEF100 the user can Import and export protocols from the IEF100 Capture data output from the IEF100 Control the IEF100 The description and screens below are HyperTerminal screens Other terminal emulator programs will also work using the same settings Configuring the Communications Port 1 Start HyperTerminal in Windows 2 Click Windows Start command 3 Launch All Programs Accessories Communications HyperTerminal 4 Assign a name to the new connection Fig 37 e p35 Serial Port Configuration 1 Select a Com Port Fig 38 This is the computer port to communicate directly with the IEF100 Choose TCP IP if connecting via the ethernet connection Fig 38 Select a Com Port TestCOM1 Properties EE Connect To Settings Country region United States Enter the area code without the long dstance prefix Area code 115 Phone number Connect using com 2 Set the baud rate to the same as the IEF100 9600 38400 or 57600 and 8 data bits no parity 1 stop bit hardware flow control Fig 39 Fig 39 Set the baud rate COM1 Properties Port Settings Bits per second Data bits 8 Parity None ei EXT m Flow control Hardware Restore Defaults e p36 TCP I
32. ctly the first time and press enter for the command to be successfully loaded into the IEF100 All commands are case sensitive Correcting typographical errors in a line of data using the backspace and delete keys will not result in successful communication The first command to type in Hyper Terminal should be AT and enter A good connection is verified with the word OK returning in the Hyper Terminal window Examples of protocols are given at the end of Appendix A These can be cut and pasted to help with the line formatting A subset of useful Hyper Terminal commands are given on the follow ing page Subset of HyperTerminal Commands Format of Command Command Description Expand Description Result of Command Command AT Check connection Check to see if the connection OK comes back AT between HyperTerminal and the with a good connection IEF100 is good and configured properly XDATALOG Transfer data Transfer data from the IEF100 Data log shows up in XDATALOG memory to HyperTerminal HyperTerminal Output Data Format Line 1 Header prot protname startdate starttime tdatapoints Line 2 datapoints minute volt uU AC1 uAC2 uAC3 Uu ACA uAC5 uACO6 temp End Line END enddate endtime Where uACI is the current in microamps in channel 1 IPROT Import protocol Import and overwrites one of the 30 Uploads protocol into 2 line formats protocol slots transfers protocol from IEF100 memory depen
33. current limit to 50 yA strip to ensure safe focusing conditions for the entire separation Note The volt and pA are scaled and graphed automatically Fig 35 Typical volts and pA profiles Typical Isoelectric Focusing The first step is typically set to low voltage to limit the current and let the ions move to the end of the IPG strips without excessive currents If loading sample with a sample cup an additional slow step may be added to allow the sample to enter the IPG strip under gentle conditions In the middle steps the voltage ramps upwards to a plateau and this is where most of the focusing occurs The current decreases down to a minimum value The graphs below show typical volt and pA profiles Fig 35 A final step has been added to the pre programmed protocols to hold the volt at 1000 V for an hour This will keep the bands focused in the IEF100 until the next step Extend the length of this step as necessary p33 Fig 36 End run screen Note Whenever a new IEF is started the previous IEF data is permanently lost Note Do not equilibrate the IPG strips before storing at 20 C e p34 Line 1 Protocol 1 7cm broad amp basic finished Line 2 6 strips Line 3 Finished at Line 4 Total 3 55 Hrs Line 5 Step 1 00 Hrs Elapsed Line 6 1000 V uA 0 1W Total 20 0 C Strip 1 Line8 13 When focusing is complete the IEF100 will beep and an end run screen will be displayed Fig 36 Line 1
34. d rs laboratoriumbruk bare Bare tilbehor og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt for drive vedlikeholde og betjene dette produktet bruker Bare en kraftforsyning som er CE merket eller sikkerhet som ha blitt sertifisert av et som nasjonalt ha blitt anerkjent prover laboratorium Sikkerheten lokket m vaere p plass for forbinding kraftforsyningene blyene til en kraftforsyning Vender all kraftforsyningsstyring av og frakopler kreftene blyene for fjerning sikkerheten lokket Sirkulerer bare vann eller 50 50 vann ethylene glykol gjennom oppvarmingen veksleren i s fall utstyrer Ikke forbind oppvarmingen veksleren til en vanntapp eller noe kjelemiddelkilde hvor vannet e piii trykket er unregulated Introduserer Aldri antifreeze eller noe organisk losemiddel inn i noe del av instrumentet Organiske losemiddler vil for rsake irreparabel skade p enheten Driver med buffertemperaturer over maksimum ikke spesifiserte teknisk spesifikasjoner overoppheting vil for rsake irreparabel skade p enheten Wazne Informacje Polish Je eli ten sprz t jest wykorzystywany w spos b nie okre lone przez Hoefer Inc do ochrony przewidzianej przez urz dzenie mo e zosta obni ony Instrument ten jest przeznaczony do u ytku w laboratoriach kryty tylko Tylko akcesori w i cz ci zatwierdzone lub dostarczone przez Hoefer Inc mog by wykorzystane do eksploatacji utrzymania i obs ugi
35. ding on computer to IEF100 step type Input Data format Delay step IPROT prot protname step delaytime delaytemp runtemp maxuA maxA maxVolt maxWatt IPROT 5 18cm const Watt D 0 00 20 20 500 12000 1 5 Step IPROT prot t protname step t steptype stepvalue timeunits timeunits time IPROT 5 18cm const Watt 2 W 1 5 V 25000 EPROT Export protocol Copies protocol number from Protocol appears in EPROT IEF100 to HyperTerminal HyperTerminal window is 1 30 EPROT Export all protocols Copies all 30 protocols from Protocol appears in EPROT IEF100 to HyperTerminal HyperTerminal window PrnRate Data interval setting Changes interval between data points INTVL in IEF100 PrnRate 1 15 minutes will update is 1 15 Start 1 2 Start protocol Starts protocol 1 with 2 IPG strips IEF100 will start focusing Start 41 42 1 is 1 30 2 is 1 6 Start Resumes protocol Restarts a protocol in a paused state IEF100 will resume focusing Start Stop Stop protocol Stops a protocol in progress IEF100 will terminate focusing Stop Pause Pause protocol Pauses a protocol in progress IEF100 will pause focusing Pause Report Sends current status Sends current run data Report if actively focusing sends current run data if paused sends a paused message if not running prints idle ID Sends serial number Transfers IEF100 serial ID number to the computer e p41 e p42 Care and Maintenance Turn the mains power sw
36. dos controles de alimentaci n y desconecta los plomos del poder antes de quitar la tapa de la seguridad Circula s lo agua o 50 50 glicol de agua etileno por el intercambiador de calor si se es el caso equiparon No conecte el intercambiador de calor a un toque de la agua ni cualquier fuente del l quido refrigerante donde la presi n del agua est libre Nunca introduce anticongelante ni alg n solvente org nico en cualquier parte del instrumento Los solventes org nicos causar n da o irreparable a la unidad No opera con temperaturas de b fer encima del m ximo especific especificaciones t cnicas Recalentar causar da o irreparable a la unidad Viktig Information Swedish om denna utrustning anv nds i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhandah ll vid utrustningen kan skadas Detta instrument formges f r inomhuslaboratorium anv ndning bara Bara medhjalpare och delar godk nde eller levererade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt anv nder bara en kraft tillg ng som r CE markerade eller s kerhet intygade vid en nationellt erk nd testande laboratorium S kerheten locket m ste vara p platsen f re koppla kraften tillg ngen blyen till en kraft tillg ng V nder sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen f re flytta s kerheten locket Cirkulerar bara vatten eller 50 50 vatten ethylene glycol
37. ebo jak koli chladic kapaliny zdroje kde tlak vody je neregulo Nikdy zav st prost edek proti zamrznut nebo jak koli organick rozpou t dla do jak koli sti z tohoto n stroje Rozpustidl m zp sob nenapraviteln po kozen jednotka Nejsou provozov na s pufru teplot ch nad maxim ln stanovenou technick mi specifikacemi P eh t zp sob nenapraviteln po kozen jednotka Vigtig Information Danish Hvis dette udstyr bruges i en m de ikke specificeret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan m ske sv kkes Dette instrument er designet for indend rs laboratoriumbrug bare Bare tilbehgr og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funktionsfejl og betjening dette produkt bruger Bare en str mforsyning der er CE markerede eller sikkerhed som er blevet attesteret af en som nationalt er blevet anerkendt prove laboratorium Sikkerhedl get m veere p plads fer forbinding stremforsyningsblyet til en str mforsyning Drejer alle stromforsyningskontroller af og afbryder kraftblyet for fjerning sikkerhedl get Cirkulerer bare vand eller 50 50 vand ethylene glykol gennem varmeveksleren i s fald udrustet Forbind ikke varmeveksleren til en vandhane eller nogen kelemiddelkilde hvor vandtrykket er unregulated Introducerer Aldrig antifreeze eller noget organisk opl sningsmiddel ind i nogen del af instrumentet Organis
38. ect the power leads before removing the safety lid Circulate only water or 50 50 water ethylene glycol through the heat exchanger if so equipped Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated Never introduce antifreeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperatures above the maximum specified technical specifications Overheating will cause irreparable damage to the unit Dulezit Informace Czech Pokud by toto za zen je pou ito zp sobem kter neni podle Hoefer Inc ochrana poskytovan na z klad za zen m e b t naru ena Tento n stroj je ur en pro vnit n pou it v laborato i pouze Pouze p slu enstv a sti schv len nebo poskytnut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku zdroj nap jen pou vaj jen e je opat en ozna en m CE osv d ena nebo bezpe nost vnitrost tn uznan mi zku ebn mi laborato Bezpe nosti lid mus b t zavedena p ed p ipojen m nap jec zdroj nap jen vede k Turn ve ker nap jen kontroly vypnuto a odpojit p ed odb rem energie vede bezpe nostn v ko Rozeslat pouze voda nebo 50 50 voda ethylenglykolu prost ednictv m v m n k tepla je li to vybavena Nemaj p ipojen v m n k tepla s vodn mi set epn n
39. er for 10 15 minutes After equilibration discard the second equilibration solution in an appropriate manner Following equilibration the IPG strips are placed on the top of the second dimension gel and sealed into place with the agarose overlay e p55 Caution SDS may cause the solution to boil over so exercise caution when heating and prevent boiling over e p56 Agarose Overlay 1 Agarose in 1X Electrophoresis Buffer Prepares 100 ml 1 agarose 25 mM Tris 192 mM glycine 0 1 SDS Prepare in a 500 ml flask to allow room for foaming Final Concentration Amount Agarose 1 lg 10X Electrophoresis Buffer 1X 10 ml 250 mM Tris 1 92M Glycine 1 SDS Bromophenol blue 3 mg Deionized water to 100 ml Gently swirl to suspend agarose Heat at low power in a microwave oven until agarose is fully dissolved Store in 1 5 ml aliquots at 4 C in plastic screw top tubes Reheat aliquots in heating block Appendix C References IEF100 Ames G E L and Nikaido K Two dimensional gel electrophoresis of membrane proteins Biochemistry 15 616 623 1976 Bjellqvist B Ek K Righetti P G Gianazza E Gorg A Westermeier R and Postel W Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6 317 339 1982 Bjellqvist B Pasquali C Ravier E Sanchez J C and Hochstrasser D A nonlinear wide range immobilized pH gradient for two dimensi
40. esis 19 758 760 1998 Rabilloud T Solubilization of proteins for electrophoretic analyses Electrophoresis 17 813 829 1996 Rabilloud T Valette C and Lawrence J J Sample application by in gel rehydration improves the resolution of two dimensional electrophoresis with immobilized pH gradients in the first dimension Electrophoresis 15 1552 1558 1994 Righetti P G Isoelectric Focusing Theory Methodology and Applications Elsevier Amsterdam 1983 Righetti P G Immobilized pH gradients theory and methodology Elsevier Amsterdam 1990 Sanchez J C Rouge V Pisteur M Ravier E Tonella L Moosmayer M Wilkins M R and Hochstrasser D F Improved and simplified in gel sample application using reswelling of dry immobilized pH gradients Electrophoresis 18 324 327 1997 Westermeier R and Naven T Proteomics in Practice A Laboratory Manual of Proteome Analysis Wiley VCH Verlag GmbH Weinheim 2002 e p58 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone 1 508 893 8999 Fax 1 508 429 5732 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc Windows and Excel are trademarks of Microsoft Corporation O 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
41. g the length of the IPG strips When using the constant watt step the units for the step value automatically change to watts In constant watt steps the IEF100 calculates the resistance and adjusts the voltage to maintain the constant watt setting Note When switching between time units the IEF100 will convert between hours and Vhrs automatically It may help to set the step time units Hrs or Vhrs before the numerical endpoint is entered Step Time Steps can be programmed to run for a set amount of time Hrs 00 00 in hours minutes or for a set amount of volt hours Vhrs volts multiplied by the number of hours Most TEF strip manufacturers recommend to run the IPG strips to a specific volt hours endpoint The researcher should determine the endpoint for their specific sample The first step with a O value entered in the step time is treated as the end of the protocol Step Type Constant Volt Gradient Volt Constant Watt Step Time Hrs hours minutes Vhrs volt hours Step Value Knob Computer Settable Settable Range Increments Increments O 12 000 250 1 O 12 000 250 1 0 1 2 0 0 1 0 1 00 00 99 59 0 15 0 01 O 300 000 500 1 e p15 Note The IEF100 instrument monitors the current and wattage in each individual strip If necessary conditions can be adjusted and or problematic strips can be removed Fig 12 Run screen Note The IEF100 also beeps between step changes e p16
42. genom varmen exchanger i s utrustad fall Inte kopplar varmen exchanger till en vatten kran eller n got kylmedel k lla d r vattnet trycket r unregulated Inf r aldrig kylv tska eller n got organiska l sningsmedel in i n gon del av instrumentet Organiskt l sningsmedel ska orsaka irreparable skada till enheten Anv nd inte med buffert temperaturer ver det h gsta angivna tekniska specifikationerna Overhettning skulle orsaka irreparabla skador p enheten piv English French German Italian Spanish Swedish La Lo La La La Waste Electrical And Electronic Equipment WEEE This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obtenir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Hausm ll entsorgt werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit eine
43. get a high pH in the sample buffer This helps solubilize some proteins and lowers enzyme activity that would attack the proteins DNA and RNA can frequently be removed by ultracentrifugation The sample can also be treated with DNase and RNase solution to break down the contaminants e Keep in mind that some protease inhibitors DNase and RNase are proteins themselves and may show up on a 2D map The water used for making reagents should be the highest quality available Water with a resistivity of gt 5 megaohm cm is best Water purified by reverse osmosis or deionized water is acceptable Recipes IEF Sample Extraction Buffer for 2D 1A Urea Sample Buffer Solution Prepares 25 ml 9 5 M urea 4 CHAPS Final Concentration Amount Urea FW 60 06 9 5M 14 26 g CHAPS 4 w v 1 0g Deionized water to 25 ml Store in 1 ml aliquots at 20 C or below Prior to use add 6 mg ml DTT to get a final sample buffer composition of 40 mM DTT Optional Add carrier ampholytes such as SERVALYTS to a concentration of 2 viv 20 ul per ml sample buffer solution 1B Thiourea Urea Sample Preparation Solution Prepares 25 ml 7 M urea 2 M thiourea 496 CHAPS 40 mM DTT Final Concentration Amount Urea FW 60 06 7M 10 51g Thiourea FW 76 12 2M 3 8g CHAPS 4 wiv 1 0g Deionized water to 25 ml Store in 1 ml aliquots at 20 C or below Prior to use add 6 mg ml DTT to get a final sample buffer composition of 40 mM DTT
44. in the top right corner of the display The run screen will display the current run conditions Note The IEF100 can only control the conditions of one focusing tray channel at a time The IEF100 limits the channel with the highest current or watts IPG strips prepared under identical conditions will show variations due to geometrical differences in the IPG gel and contact differences under the electrodes IPG strips with different samples can show large differences in current and wattage due to different conductivities of the samples Fig 33 Run screen Line 1 Line 2 Line 3 Line 4 Line 5 Line 6 Line 8 13 Run Screen The run screen displays all the information on the current run Fig 33 There are no editable fields The information on the run screen is described below Line 1 The protocol number and name are shown The top right corner indicates the run status as either running flashing paused or finished Line 2 Displays the number of IPG strips being focused or number of channels if using the Dual Electrode Accessory Line 3 Describes the active step the step number the step value volt or watt strip and the step end point Hrs or Vhrs Line 4 Displays the total elapsed time and the total volt hours of focusing up to the moment Line 5 Displays the total elapsed time and total volt hours of the active step Line 6 Displays the real time output conditions of all the strips including the cu
45. ined automatically NO CONNECTION will be displayed until an ethernet cable is attached to the port Once an active LAN connection is established the local server will assign an IP address that will be displayed in the XXX XXX XXX XXX format This process usually takes about 10 seconds e p17 p18 To Set the Date The date field is three separate fields of DAY MONTH YEAR 1 2 Use the knob to highlight the date field Click the knob to set the date Turn the knob to change the date Use the horizontal arrows the keys directly beneath the knob to access the MONTH and YEAR fields if necessary When all three fields are correct press the knob to enter the new date To Set the Time The time field is two separate fields HOUR MINUTE The HOUR field uses the range 0 24 to denote the time of day 1 Use the knob to highlight the time field Click the knob to set the time 2 Turn the knob to change the HOUR Use the horizontal arrows the keys directly beneath the knob to access the MINUTE field if necessary 3 When both fields are correct click the knob to enter the new time Diagnostics 1 Highlight diagnostics and click the knob The function key RUN appears on the screen Press RUN to show a popup screen with three options Manual Vout 2 Use the knob to select the option and press the function OK Run Diags The diagnostics function has the IEF100 go through a series of inter nal tests to confirm functio
46. itch off and unplug the power cord before cleaning Instrument and Lid Use a soft cloth dampened with water or a mild cleaning solution to clean the cabinet and display e Tf spilled liquids contact the circuit boards unplug the IEF100 and allow to dry completely Call Hoefer Inc for advice before using Cleaning the Tray and Electrodes detergent designed to remove oil is recommended followed by mild nonionic laboratory cleaning reagents Rinse tray well with deionized water and dry completely before use The platinum wire on the electrodes is fragile and should be cleaned very gently The electrodes should not be soaked in solution Technical Service and Repair Hoefer Inc offers complete technical support for all of our products If you have any questions about how to use this product or would like to arrange to repair it please call or fax your local Hoefer Inc representative Troubleshooting Problem Solution Instrument does not turn on Check power cord is plugged into grounded outlet and ON OFF switch is turned to the on I position Lid is open Unit will not operate unless the safety lid is fully closed Press down on both sides to engage latches Unit reads O pA O W for all strips Ensure the electrode wicks are in contact with the IPG strips The electrodes should be contacting the electrode wicks and centered in the area of overlap between the wick and the IPG strips
47. ke opl sningsmidler vil for rsage uboelig skade til enheden Driver ikke med st dpudetemperaturer over maksimummet specificerede tekniske specifications Overheding vil for rsage uboelig skade til enheden Belangrijke Informatie Dutch Indien deze uitrusting in een manier wordt gebruikt die niet door Hoefer Inc is gespecificeerd de bescherming die door de uitrusting is verzorgd kan worden geschaad Dit instrument is voor binnenlaboratoriumgebruik enkel ontworpen Enkel onderdelen en delen keurden goed of leverden door Hoefer Inc kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product gebruik Enkel een netvoeding die CE is markeerde of veiligheid die door een is gecertificeerd die nationaal is herkend testene laboratorium Het veiligheidsdeksel moet in plaats voor het verbinden van de netvoeding leidt tot een netvoeding zijn Doe alle netvoedingscontroles Uit en koppel los de machtleiding voor het verwijderen van het veiligheidsdeksel Circuleer enkel water of 50 50 water ethyleenglycol door de hitte exchanger zo ja uitrust Verbind de hitte exchanger naar een waterkraan of koelmiddelbron niet waar de waterdruk niet geregulariseerd is Stel Nooit antivriesmiddel of organische oplosmiddelen in deel van het instrument voor Organische oplosmiddelen zullen onherstelbare schade aan de eenheid veroorzaken Bedien niet met buffertemperaturen boven het maximum specificeerde technische specificaties
48. m autorisierten Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este s mbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el equipo Denna symbol anger att elektriska och elektroniska utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information ang ende avyttring av utrustningen e pv Note Ohm s law states that the 10 mA of output is available at 1000 V and less Fig 1 The Hoefer IEF100 Function and Description The Hoefer IEF100 Isoelectric Focusing instrument is designed to perform isoelectric focusing of proteins in Immobilized pH Gradient IPG strips It is an integrated system with a built in Peltier cooled temperature platform and a 12 000 V 10 W power supply capable of delivering up to 10 mA There are 30 fully editable prot
49. mani re pas sp cifi par Hoefer Inc la protection fourni par l quipement pourrait tre diminu e Cet instrument est concu pour l usage de laboratoire int rieur seulement Seulement les accessoires et les parties ont approuv ou ont fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entretenir ce produit utilise Seulement une alimentation qui est CET a marqu ou la s curit certifi par un nationalement reconnu essayant le laboratoire Le couvercle de s curit doit tre sa place avant connecter l alimentation mene une alimentation Tourner tous contr les d alimentation de et d brancher les avances de pouvoir avant enlever le couvercle de s curit Circuler seulement de l eau ou 50 50 glycol d eau thyl ne par l exchanger de chaleur si si quip Ne pas connecter l exchanger de chaleur un robinet d eau ou la source d agent de refroidissement o la pression d eau est non r gul e Ne Jamais introduire d antigel ou du dissolvant organique dans n importe quelle partie de l instrument Les dissolvants organiques causeront des dommages irr parables l unit Ne pas fonctionner avec les temp ratures de tampon au dessus du maximum a sp cifi des sp cifications techniques La surchauffe causera des dommages irr parables l unit Wichtige Informationen German Wenn diese Ausr stung gewisserma en nicht angegeben durch Hoefer Inc verwendet wird kann de
50. mscheit A and G nther S Methodology of two dimensional electrophoresis with immobilized pH gradients for the analysis of cell lysates and tissue proteins in Endler AT and Hanash S eds Two Dimensional Electrophoresis Proceedings of the International Two Dimensional Electrophoresis Conference Vienna Nov 1988 VCH Weinheim FRG 1989 e p57 E BEBES G rg A Obermaier C Boguth G Posch A and Weiss W Two dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension IPG Dalt the state of the art and the controversy of vertical vs horizontal systems Electrophoresis 16 1079 1086 1995 G rg A Obermaier C Boguth G Csordas A Diaz J J Madjar J J Very alkaline immobilized pH gradients for two dimensional electrophoresis of ribosomal and nuclear proteins Electrophoresis 18 328 337 1997 Link A J ed 2 D Proteome Analysis Protocols Methods in Molecular Biology 112 Humana Press 1998 O Farrell P H High resolution two dimensional electrophoresis of proteins J Biol Chem 250 4007 4021 1975 Olsson I Larsson K Palmgren R and Bjellqvist B Organic disulfides as a means to generate streak free two dimensional maps with narrow range basic immobilized pH gradient strips as first dimension Proteomics 2 1630 1632 2002 Rabilloud T Use of thiourea to increase the solubility of membrane proteins in two dimensional electrophoresis Electrophor
51. n The tests include the voltage and current output of the internal high voltage power supply the eprom and the clock If everything tests OK the message All OK is briefly displayed on the screen If one of the tests fail contact the Hoefer Inc representative to arrange service for the instrument Manual Vout Press OK to show a run screen Click the knob to SET mode and the voltage output is adjustable in 250 V increments The manual run will continue until stopped by the user or up to 4 hours Exiting this screen will return to the main menu PA Clear All Protocols This option is used to overwrite the existing protocols in memory A Note You cannot recover user popup screen shows three options programmed protocols once they have been reset or erased Defaults Erase Cancel e Highlight the appropriate option and press OK e DEFAULTS resets all 30 protocols to the factory default settings e the nine pre programmed protocols and 21 empty protocols e ERASE deletes all the data in all 30 protocols in memory Empty protocols have default limits of 12 000 V 500 uA strip 2 W strip and 20 C Serial Number Displays the serial number of the IEF100 This is not editable e p19 Note Push the knob inwards and release quickly to click There is tactile and audible feedback A successful click changes between MOVE and SET modes MOVE SET Q e Fig 14 The knob Fig 16 Ethernet
52. not equilibrate the IPG strips before storing at 20 C 3 SDS Equilibration Buffer Solution This solution is used after IEF and before second dimension PAGE The IPG strips are immersed in excess solution to raise the pH of the strip buffer so that it is suitable for PAGE and to coat the proteins in SDS uniformly so that they migrate properly in the second dimension gel Prepares 200 ml 6 M urea 75 mM Tris HCI pH 8 8 29 3 glycerol 2 SDS 0 002 bromophenol blue Final Concentration Amount Urea FW 60 06 6M 72 1 1 5M Tris HCl pH 8 8 stock solution 75 mM 10 0 ml Glycerol 87 w w 29 3 v v 69 ml SDS FW 288 38 2 wiv 408 Bromophenol blue 0 002 w v 4 mg Deionized water to 200 ml Aliquot into 30 ml aliquots and store frozen at 20 C or below 24 cm IPG s require 5 10 ml per strip per equilibration step Shorter strips can use proportionately less volume per equilibration step Equilibration Procedure 1 Thaw two aliquots of the equilibration solution Add 10 mg ml DTT to one solution Place the IPG strips in the rehydration equilibration tray Add 6 5 ml of solution to each slot containing an IPG strip ao 5 W N Place on rocker for 10 15 minutes After equilibration discard the first equilibration solution in an appropriate manner 6 X Add 25 mg ml lodoacetamide IAA to the second aliquot of equilibration solution Add 6 5 ml of solution to each slot containing an IPG strip 8 Place on rock
53. ns screen is accessed the IEF100 will first check to see if there is an ethernet connection The IEF100 is inaccessible until the check is completed about 10 seconds The words Checking Ethernet will flash until the check is completed Fig 13 Options screen Options Screen From the main menu pressing the OPTION key will allow access to the following options Fig 13 Serial Port and Intvl Ethernet Date Time Diagnostics Clear all protocols Serial Number A Agioeter IEF100 oe Options Serial Port 38400 Intvl 5 min Ethernet NO CONNECTION Date Time dd mm yyyy hh mm Diagnostics Clear all protocols Serial Number xxxxxxxx HIGH O VOLTAGE MAIN RUN OOOO IO eI J Serial Port The serial port sets the speed at which data is sent to or received from an external device The baud rate can be set to 9600 38400 or 57600 with 38400 being the default The baud rate should match the data reporting rate of the external device computer or printer serial port Intvl The time interval between data points sent to an external receiv ing device The IEF100 internally records data points every minute When downloading the data the interval between data points can be changed from 1 to 15 minutes The default interval is 5 minutes For long runs it may be desirable to have fewer data points and therefore use higher values for the interval Ethernet The ethernet connection is determ
54. ocol 5 narrow D 0 00 20 20 500 12000 1 5 narrow 1 G 1000 H 1 00 narrow 2 G 12000 H 1 00 narrow 3 S 12000 V 50000 narrow 4 S 1000 H 1 00 narrow 5 S 0 H 0 00 narrow 6 S 0 H 0 00 narrow 7 S 0 H 0 00 narrow 8 S 0 H 0 00 narrow 9 S 0 H 0 00 IPROT 5 18cm const Watt D 0 00 20 20 500 12000 1 5 IPROT 5 18cm const Watt 1 W 0 1 H 1 00 IPROT 5 18cm const Watt 2 W 1 5 V 25000 IPROT 5 18cm const Watt 3 S 1000 H 1 00 IPROT 5 18cm const Watt 4 S 0 H 0 00 IPROT 5 18cm const Watt 5 S 0 H 0 00 IPROT 5 18cm const Watt 6 S 0 H 0 00 IPROT 5 18cm const Watt 7 S 0 H 0 00 IPROT 5 18cm const Watt 8 S 0 H 0 00 IPROT 5 18cm const Watt 9 S 0 H 0 00 Protocol 6 IPROT 6 24cm broad amp basic D 0 00 20 20 500 12000 2 0 IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm IPROT 6 24cm broad amp basic 1 G 1000 H 1 00 broad amp basic 2 G 12000 H 1 00 broad amp basic 3 S 12000 V 45000 broad amp basic 4 S 1000 H 1 00 broad amp basic 5 S 0 H 0 00 broad amp basic 6 S 0 H 0 00 broad amp basic 7 S 0 H 0 00 broad amp basic 8 S 0 H 0 00 broad amp basic 9 S 0 H 0 00 e p49 e p50 Protocol 7 IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm IPROT 7 24cm Protocol 8 narrow D 0 00 20 20 500 12000 2 0 narrow 1 G 1000 H 1 00 narrow 2 G 12000 H 1 00 narrow 3 S 12000 V 100000 narrow 4 S 1000 H 1 00 narrow 5 S 0 H
55. ocols nine of which are pre programmed with recommended IEF protocols One running tray will hold all currently available IPG strips 3 0 3 5 mm wide The IPG strips are run gel side up using electrode wicks to help remove impurities that collect at the end of the pH gradients Samples can be loaded into the IPG strips during rehydration or with sample cups The IEF100 is fully tested and certified to all applicable international standards Features of the IEF100 are One 6 channel tray that focuses Up to 6 7 24 cm IPG strips using the single electrode set Up to 12 7 cm IPG strips using the dual electrode accessory included Sample loading during rehydration or with sample cup Monitors current in each IPG strip Integrated 12 000 volt DC power supply with 10 mA capability the most voltage and current capability available A large blue white LCD display for easy viewing that supports a graphical display of voltage and current The ability to program edit and store up to 30 protocols each with 9 steps Constant power mode e A real time clock settable to local time zones e Temperature controlled platform Ethernet and RS232 ports to report data or load protocols e pl This declaration of conformity is only valid for the instrument when it is used in laboratory locations e used as delivered from Hoefer Inc except for alterations described in the user manual and e connected to othe
56. on method Load more protein or try a more sensitive method of detection IPG strip was not properly hydrated Ordering Information Product Quantity Order Code IEF100 1 IEF100 Running Tray 1 IEF105 Electrode Wicks 504 IEF106 Sample Loading Cups 10 strips of 6 60 1EF108 Small Rehydration Equilibration Tray 1 IEF109 Medium Rehydration Equilibration Tray 1 IEF111 Large Rehydration Equilibration Tray 1 IEF110 Power Cord US 115 V 1 PSCORD 115V Power Cord Euro 230 V 1 PSCORD 230V Second Dimension Units Mighty Small Il Deluxe Mini Vertical Unit SE260 10A 1 5 Deluxe Dual Cooled Standard Vertical Unit SE600X 15 1 5 Large Format Vertical Unit SE900 1 0 Reagents Agarose 500 g GR140 500 Bromophenol Blue Sodium Salt 10 g GR120 10 CHAPS 10g GR121 10 Dithiothreitol DTT 5g GR122 5 Glycerol LE GR124 1 Mineral Oil 1L GR138 1 Sodium Dodecyl Sulfate 500 g GR126 500 Thiourea 500 g GR130 500 Tris 1 kg GR132 1 Urea 1 kg GR143 1 ep45 e p46 Appendix A Preprogrammed Protocols The following 9 protocols are preprogrammed They are to be used as guidelines Each protocol has a 1 hour 1000 V hold step to maintain sharp bands once focusing is complete This step is not necessary and can be removed It can also be extended as desired Program 1 Focusing of most 7 cm IPG s by limiting volt parameters Name 7 cm broad amp basic Delay 0 00 Delay temp 20 C Run temp 20 C 500 pA 6000 V 0 5 W Step 1 Gradient volt 500 V
57. onal electrophoresis and its definition in a relevant pH scale Electrophoresis 14 1357 1365 1993 Dunn M J and Corbett J M 2 dimensional polyacrylamide gel electrophoresis Methods Enzymol 271 177 203 1996 Eckerskorn C Jungblut P Mewes W Klose J and Lottspeich F Identification of mouse brain proteins after two dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis Electrophoresis 9 830 838 1988 Gorg A Postel W G nther S The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 9 531 546 1988 Gorg A Postel W Weser J G nther S Strahler J R Hanash S and Somerlot L Elimination of point streaking on silver stained two dimensional gels by addition of iodoacetamide to the equilibration buffer Electrophoresis 8 122 124 1987 Gorg A Two dimensional electrophoresis Nature 349 545 546 1991 Gorg A Obermaier C Boguth G Harder A Scheibe B Wildgruber R and Weiss W The current state of two dimensional electrophoresis with immobilized pH gradients Electrophoresis 21 1037 1053 2000 Gorg A Obermaier C Boguth G and Weiss W Recent developments in two dimensional gel electrophoresis with immobilized pH gradients wide pH gradients up to pH 12 longer separation distances and simplified procedures Electrophoresis 20 712 717 1999 Gorg A Postel W Do
58. onstant Volts 8 Constant Voltst 9 Constant Volts MAIN OOOO Use the knob to move between the following editable fields Once highlighted click the knob to change the value of the field Protocol Number Click the knob The knob will now cycle through the 30 protocols allowing a quick review of each protocol Protocol Name Protocol names can have up to 16 alphanumeric symbols The following symbols are available ABCDEFGHIJKLMNOPQRSTUVWXYZ 96 9 mun 0123456789 Highlight the protocol name field and click the knob A cursor will cause the first character to blink Turn the knob to scroll through the above symbols Once the desired symbol is reached use the horizontal arrows the keys directly beneath the knob to move the cursor to the next position Repeat until the protocol name is correct The blank space is between the lower case n and the 0 symbols Note Times to the nearest minute increment can be uploaded to protocols from a computer Note It is recommended not to exceed 6000 V or more than 0 5 W strip for a 7 cm IPG Please follow the IPG strip manufacturer s instructions for recommended limits 33 66 45 The lower case m u and n can be used to designate milli micro and nano if desired When the name is correct click the knob to enter the name and move to the next field Delay The delay line is an optional step Both a time and a
59. p can be removed and the remaining strips will focus at higher voltages 7 cm strips should not be focused at volts higher than 6000 V and watts higher than 0 5 W per strip Browning burning of strips arcing erratic pA readings Strips have high conductivity due to salts or other ionic compounds Reduce the ionic strength by limiting salt concentration to 10 mM or below Reduce Tris concentrations to 50 mM or below This indicates the power volt or current delivered to the strip is too high Limit current in the IPG strips to DO pA Too little oil and or strips not completely covered in oil Level the instrument Ensure mineral oil completely covers the strips and the empty channels If strips are not completely covered urea will crystallize out and cause local heating burning Overheating of the tray Check tray is securely slid and locked into place to ensure the best contact between the tray base and the platform surface Rehydration equilibration tray has residual liquid after rehydration of IPG strips Excess volume used Do not exceed the IPG strip manufacturer s recommended volumes Strip accidentally rehydrated with the gel side up or strip cover sheet not removed Extend rehydration time 8 10 hours minimum is required for the best uptake of rehydration solution No proteins present in gel after 2nd dimension completed e p44 Load of proteins is too little for the detecti
60. r CE labeled instru ments or products recommended or approved by Hoefer Inc e p2 Specifications Capacity using Single Electrode set Capacity using Dual Electrode accessory Output Power in User interface Platform temperature Protocol capacity Input output ports Dimension w x d x h Weight Environmental operating conditions Product certifications 1 6 IPG strips 7 to 24 cm long 2 12 IPG strips 7 cm long Volt 12 000 V Current 10 mA Power 10 W 100 240 V 50 60 Hz 100 W Large blue white graphical display Knob 7 button keypad 15 25 C 30 programs with 9 steps each Ethernet RS232 38 cm x 27 cm x 19 cm 8 kg Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category II Pollution degree 2 EN61010 1 2001 EN61326 1998 CE WEEE RoHS Fig 2 The IEF100 unit System Components Unpacking Unwrap all packages carefully and compare the contents with the packing list making sure all items arrived If any part is missing contact your local Hoefer Inc sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Required but not supplied Immobilized pH Gradient IPG strips Reagents required for sample preparation and strip rehydration
61. r durch die Ausr stung zur Verf gung gestellte Schutz verschlechtert werden Dieses Instrument wird f r den Innenlaborgebrauch nur daf r entworfen Nur Zus tze und Teile genehmigten oder lieferten durch Hoefer Inc kann f r das Funktionieren das Aufrechterhalten und die Wartung dieses Produktes verwendet werden Verwenden Sie nur eine Energieversorgung die CE gekennzeichnet oder durch ein national anerkanntes Probelaboratorium bescheinigte Sicherheit ist Der Sicherheitsdeckel muss im Platz vor dem Anschlie en der Energieversorgung sein f hrt zu einer Energieversorgung Alle Energieversorgungssteuerungen abdrehen und die Macht trennen f hrt vor dem Entfernen des Sicherheitsdeckels Nur Wasser oder 50 50 Glykol des Wassers Athylens durch den W rmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder K hlmittel Quelle nicht wo der Wasserdruck ungeregelt wird F hren Sie nie Frostschutzmittel oder jedes organische L sungsmittel in jeden Teil des Instrumentes ein Organische L sungsmittel werden nicht wiedergutzumachenden Schaden der Einheit verursachen Mit Puffertemperaturen ber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die berhitzung wird nicht wiedergutzumachenden Schaden der Einheit verursachen Informazioni Importanti Italian Se quest apparecchiatura usata in un modo specificato da Hoefer Inc
62. rall ionic strength Some protein samples will readily solubilize while others are more difficult requiring additional reagents such as thiourea special detergents etc to encourage solubilization The common classes of additives are listed below Denaturants Urea is the most common reagent used in IEF for disrupting the internal bonding of the protein allowing it to unfold Samples are prepared using urea concentrations from 8 9 5 M In general the higher the urea concentration the better a sample can be solubilized Urea reaches its saturation point near 10 M at room temperature Thiourea is also used to better solubilize some samples Frequently 2 M thiourea is combined with 5 7 M urea as a reagent for sample prep and IPG rehydration Detergents Several types of non ionic or zwitterionic detergents can be used to solubilize samples CHAPS Triton X100 Nonidet NP 40 and alkylamidosulfobetaine detergents CHAPS is the most widely used detergent for 2D electrophoresis It is stable in solution Detergents such as SDS are not compatible with IEF because they bind to the proteins and mask the proteins native charge Reductants Dithiothreitol DTT is commonly used to reduce proteins in IEF DTT breaks down in solution so it is normally prepared and added just before use Other reductants such as 2 Mercaptoethanol Dithioerythritol DTE and Tributylphosphine TBP can be used Other Carrier ampholytes or IPG buffers
63. real time Fig 16 See Optional Data Connections page 35 Preparing IPG Strips Immobilized pH Gels IPG are ultra thin polyacrylamide gels on plastic support films The gels are made with a pH gradient covalently bonded into the gel matrix The gels are stable and reproducible The gels are supplied dehydrated and should be stored at temperatures 20 C or below The IPG strips have an acidic end and a basic end 2D gel images are typically shown with the acidic side on the left and that is the orientation of the IPG strips in the IEF100 The proper electrode must be applied to each end anodic electrode to the acidic end left and the cathodic electrode to the basic end right The dried strips require rehydration before the run Typically several different reagents are included in the rehydration solution to help solubilize the proteins and enable successful isoelectric focusing Rehy dration is typically done at room temperature or 20 C and needs a minimum of 8 hours for good absorption of the reswelling solution It is frequently more convenient to allow rehydration to occur overnight During rehydration the IPG strips are covered in mineral oil to prevent loss of moisture and prevent urea crystallization The IPG strips are rehydrated using one of the rehydration equilibra tion trays supplied with the IEF100 Protein sample can be added to the IPG strip in this step The IPG strips are transferred to the IEF1
64. rrent volts the total current the total watts and the platform temperature Line 8 13 Display the individual strip current and watts Hoefer MOVE Alt EF100 S Ful Protocol 7 24cm narrow running 6 strips Step 3of4 5w strip 100 000 Vhrs Total 4 24 19108 Vhrs Step 1 02 12088 Vhrs Elapsed 11574V 156uA 1 8 W Total 20 0 C Strip 26 uA 0 3 W 28 uA 0 3 W 35 uA 0 4 W 24 uA 0 3 W 17 uA 02W HIGH O VOLTAGE 26 uA 0 3 W FER MAIN PAUSE NEXT GRAPH e p31 Fig 34 Run screen p32 In the example below 6 IPG strips are being focused according to protocol 7 a maximum of 0 5 W strip and 100 000 volt hour endpoint Fig 34 The protocol is in the third step The protocol has been running for 4 hours and 24 minutes or 19 108 volt hours Currently the protocol is 1 hour and 2 minutes and 12 088 volt hours into step 3 The internal power supply is delivering 11 574 volts a total of 156 pA and 1 8 watts across six IPG strips During a run different strips may control the overall voltage as the resistance of the strips changes during the run Protocol 7 24cm narrow running 6 strips Step 3of4 0 5 Wistr 100 000 Vhrs Total 4 24 Vhrs Step 102 20 Elapsed 11574V 156uA 1 8 W Total 20 0 C p OA PSU PS sa sa sas Note Extra salt in the sample tends to flatten the graphs horizontally and take longer for the sample to focus Note If there are problems with burning IPG strips set the
65. strips with a protective cover If present peel off protective layer Place the strip gel side down onto the liquid in the channel Gently slide the strip back and forth to wet the entire length of the IPG gel with rehydration solution Sometimes lifting and lowering the IPG strip can aid in getting the proper contact of the solution over the entire length of the IPG gel strip Cover the strips with mineral oil Hoefer order number GR138 1 Allow to rehydrate a minimum of 8 hours Better rehydration occurs if allowing strips to rehydrate overnight Fig 18 Slide the focusing tray Fig 19 Load the IPG strips Note In any one run all the IPG strips should be the same length for the electrodes to make good contact Isoelectric Focusing using the Single Electrode Set Open the safety lid by pressing down on the white push label in the middle of the front of the lid Fig 17 o Insert the IEF105 focusing tray on the far right side of the cold plate The tray has only one orientation There are and marks on the tray that align with corresponding marks on the IEF100 The tray will not fit properly in the IEF100 in any other orientation e Slide the focusing tray to the left under the clamping tabs Fig 18 These tabs improve the contact and the heat transfer between the tray and the cold plate o Use forceps to load the rehydrated IPG strips into the focusing tray gel side up The anodi
66. te reconhecido testando laborat rio A tampa de seguran a deve estar em lugar antes de ligar o estoque de poder leva a um estoque de poder Desliga todos controlos de estoque de poder e desconecta os chumbos de poder antes de retirar a tampa de seguran a Circulam s gua ou 50 50 glicol de gua ethylene pelo exchanger de calor se for assim equiparam N o ligue o exchanger de calor a uma torneira de gua nem qualquer fonte de refrigerante onde a press o de gua n o regulado Nunca introduz anticongelante nem qualquer org nico solvente em qualquer parte do instrumento Org nico solvente causar agress o irrepar vel unidade N o opera com temperaturas de buffer acima do m ximo especificou especifica es t cnicas Superaquecer causar agress o irrepar vel unidade Informaci n Importante Spanish Si este equipo es utilizado en una manera no especificado por Hoefer Inc la protecci n proporcionado por el equipo puede ser da ada Este instrumento es dise ado para el uso interior del laboratorio s lo S lo accesorios y partes aprobaron o suministraron por Hoefer Inc puede ser utilizado para operar para mantener y para atender a este producto S lo utiliza una alimentaci n que es CE marc o la seguridad certificada por un nacionalmente reconocido probando el laboratorio La tapa de la seguridad debe estar en el lugar antes de conectar la alimentaci n lleva a una alimentaci n Apaga to
67. to one column in an Excel spreadsheet Use the Excel menu Data Text to columns command set the delimiter as commas and the data will sort into columns for graphing Capturing IEF100 Data 1 Make sure a good connection exists between HyperTerminal and the IEF100 Open HyperTerminal Type AT and press ENTER OK should appear in the HyperTerminal window There are two ways to download data from the IEF100 The data can be collected in real time or sent as a data log file after focusing Collect in Real Time 1 With HyperTerminal program open use the HyperTerminal menus to access Transfer Capture text Fig 44 Name the file and directory that will collect the data Use the browse button if necessary Fig 45 The data from the IEF100 is sent automatically to the output ports every 5 minutes Once a run is started data will automatically start appearing in the HyperTerminal window After focusing to stop the data capture use the menus Transfer Capture Text Stop or Pause Fig 46 The data is stored in the named file Transfer Data Log After IEF 1 2 Type XDATALOG in an active HyperTerminal window The data from the last run will be transferred to the HyperTerminal window e p39 e p40 HyperTerminal Commands The Hyper Terminal program communicates with the IEF100 by trans mitting one line of data at a time Multiple lines can be typed and sent together Type each line perfe

IEF100 User Manual – English

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IEF100 User Manual &ndash; English

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IEF100 User Manual – English