IPGphor Isoelectric Focusing System User Manual
Contents
1. fe Step n hold OV 0 00 Hrs The two options for the voltage change pattern are Step n Hold which sets the voltage at the selected value for the new step and then holds the voltage con stant for the step duration and Gradient which increases the voltage limit lin early with respect to time from the value set for the previous step to the value set for this step If this is the first step the value increases from 0 The original programmed option is Step n Hold for each step To toggle to Gradient move the cursor to the Step n Hold field using the right and left arrow keys and toggle with the up or down arrow key 1 tep n hol N OV 0 Hrs 1 Gradient OV 0 00Hrs IEF voltage is programmable from 0 8000 V in increments of 10 V Each of the first three digits in the voltage field can be set independently First move the cur sor to the desired digit location using the right and left arrow keys and then change the value using the up and down arrow keys LT 1 Step n hold 500 0 00Hrs The original factory programmed voltage for each step is 0 Program each step with the protocol voltage values If fewer than nine steps are required end the program by assigning a value of O V after the final step The step duration 0 to 99 59 hours or volt hours 0 to 99999 Vhr are program mable for each step To select set the cursor under the currently selected field and use the up2. Be sure to keep all packing material for damage claims or to use should it become nec essary to return the unit 1 2 Instrument components The mains power switch is located on the back panel on the left side Negative cathode elec trode area Control panel Five strip holder lengths are available 7 11 13 18 and 24 cm one for each IPG strip length Cover Pressure block Note All strips and holders used at any one time must be of the same length Base Sample application wells o Control panel COMMAND heys HV ON indicator lamp Platform Electrode areas Safety lid Strip holders Fixed length strip holders Sample application wells Cover Cup loading strip holder Electrodes Sample cups Cover The IPGphor is controlled by a 7 key membrane keypad Instrument status is indicated on the LCD screen LCD screen The main screen is Protocol 1 Name The default location of the cursor is always under the protocol number 1 Select any protocol by pressing the up and down arrow keys to display the number of the desired protocol Once the selected protocol is started the LCD screen indicates the parameters of the step currently being carried out In edit mode the LCD displays the programmable parameters Place the cursor in the field to be edited using the right and left arrow keys and adjust the value by pressing the up and down arrow keys The E
3. Programmable power parameters include maximum current voltage and voltage change pattern Please refer to the 2 D Electrophoresis principles and methods guide for sample han dling recommendations and protocol guidelines Protocol worksheets pages 26 28 can aid in mapping out and documenting each protocol The allowable range of values for each programmable parameter is listed below Parameter operational range recommended values Steps 1 9 Step duration hrs rehydration 0 99 59 10 00 14 00 focusing 0 99 59 2 00 10 00 Volts V 0 8000 100 8000 Max current 1500 pA total for 12 strips max 50 pA per strip Platform temperature C rehydration 18 25 20 focusing 18 25 20 Navigating parameter fields and digits Each screen consists of information as well as one or more fields that can be edited The active fields contain individually adjustable digits or characters Certain digits are also linked to their neighboring dig its For example three of the four digits in the voltage field can each be adjusted indi vidually but when adjusting the value from 1000 to 990 for instance even though only the ten s place is adjusted manually three digits are affected Whether individual or linked digits wrap around so increasing from 9 produces 0 and decreasing from 0 produces 9 until a parameter limit is reached Screen 1 Rehydration parameters 10 3 2 2 Editing protocol parameters All protocol parameters
4. 24 cm complete 1 80 6470 07 Cup Loading 3 80 6459 43 Immobiline DryStrip Reswelling Trays 7 18 cm 1 80 6371 84 7 24 cm 1 80 6465 32 IPG Buffer pH 4 7 1 ml 17 6000 86 pH 3 10 L 1 ml 17 6000 87 pH 3 10 NL 1 ml 17 6000 88 pH 3 5 5 0 1 ml 17 6002 02 pH 4 5 5 5 1 ml 17 6002 04 pH 5 0 6 7 1 ml 17 6002 05 pH 5 5 6 7 1 ml 17 6002 06 pH 6 11 1 ml 17 6001 78 PlusOne reagents Urea 500 g 17 1319 01 CHAPS 18 17 1314 01 Triton X 100 500 ml 17 1315 01 Dithiothreitol DTT 18 17 1318 01 Bromophenol Blue 10g 17 1329 01 Immobiline DryStrip Cover Fluid 1000 ml 17 1335 01 25 26 Immobiline DryStrip gels 7cm pH 4 7L pH 3 10L pH 3 10NL pH 6 11NL 11cm pH 4 7L pH 3 10L pH 6 11NL 13 cm pH 4 7L pH 3 10L pH 3 10NL pH 6 11NL 18cm pH 4 7L pH 3 10L pH 3 10NL pH 3 5 4 5 pH 4 0 5 0 pH 4 5 5 5 pH 5 0 6 0 pH 5 5 6 7 pH 6 9 pH 6 11 pH 3 5 4 5 pH 4 0 5 0 pH 4 5 5 5 pH 5 0 6 0 pH 5 5 6 7 pH 3 7 NL pH 3 10 Cleaning solution IPGphor Strip Holder 2D Electrophoresis Principles and Methods Equilibration tube set for 24 cm IPG Strips IEF electrode strips Code No 17 6001 10 17 6001 11 17 6001 12 17 6001 94 18 1016 60 18 1016 61 17 6001 95 17 6001 13 17 6001 14 17 6001 15 17 6001 96 17 1233 01 17 1234 01 17 1235 01 17 6001 83 17 6001 84 17 6001 85 17 6001 86 17 6001 87 17 6001 88 17 6001 97 17 6002 38 17 6002 39 17 6002 40 17 6002 41 17 6002 42 17 6002 43 17 6002 44 17 6002 45 17 6002 46 17 60
5. 5 4 Printing a log of instrument conditions The RS232C serial port is programmed to report instrument status every 5 minutes during isoelectric focusing The rehydration period is not logged Indicated are the time elapsed voltage current and cumulative volt hours Also indicated are any inter ruptions in power such as when the lid is opened or when the end of the programmed run is reached An abbreviated log illustrates the format 21 Important You must use a neutral pH detergent such as the IPGphor Strip Holder Cleaning Solution to remove residual protein from these strip holders Important Strip holders may be baked boiled or autoclaved DO NOT EXPOSE THEM TO STRONG ACIDS OR BASES INCLUDING ALKALINE DETERGENTS 22 Care and Maintenance 4 1 Important Strip holders are made of ceramic and thus are very brittle and fragile Handle carefully Do not bump or drop When cleaning take care not to bend or break the strip holder electrodes Take care to avoid scratching the electrode areas m Wipe the exterior surfaces with a damp cloth after each use Never use abrasive cleansers or solvents Always turn the mains power switch off and unplug the power cord before doing any maintenance 4 2 Cleaning The parts of the instrument that come in contact with liquid reagents are resistant to chemicals typically used for IEF Before introducing any other chemicals into the system first test the affec
6. C 0 90 noncondensing 115 230 V 50 60 Hz 100 W 0 8000 V resolution 10 V 0 1 5 mA resolution 0 01 mA 12 W max 14 x 25 x 46 cm 6 8 kg 15 lbs Automatic voltage cutoff when safety lid is opened CE UL CSA This declaration of conformity is only valid for the instrument when it is D used in laboratory locations and D used as delivered from Amersham Pharmacia Biotech except for alterations described in the User Manual Instrument Set Up 2 1 Placing the instrument 1 Important The vents at the front back and bottom of the instrument must not be obstructed Air must be free to circulate for the cooling apparatus to function properly 2 Place the instrument on a level surface to prevent liquid from draining out of the strip holders 2 2 Power connection Two power cords are supplied one cord has a North American style NEMA 5 15 P 3 pronged plug for 115 V power outlets and the other has a Central European style CEE 7 7 plug for 230 V power outlets 1 Select the cord with the appropriate plug for the power outlet in your laboratory If neither of these cords are suitable for your power outlets obtain a detachable cord with an IEC 320 C13 input 2 Plug the connectors into the power cord receptacle on the rear panel and into a properly grounded power outlet 2 3 Optional Serial port connector for a printer or computer connection The RS232C serial port for a printer or compute
7. after each use Other exterior surfaces can be cleaned with a damp cloth Never use abrasive cleanser or solvents 4 3 Replacing fuses Important Fuses protect equipment by disconnecting loads too large for the instru ment s circuit design so it is imperative that fuses are replaced only vvith fuses of iden tical rating The mains power module located at the back of the instrument contains two input fuses which are the same for all models T 1 6A 250V 5 x 20 mm Insert screwdriver in this notch to open the cover The mains power module is located on the back panel Mains power switch Insert the screwdriver blade behind the arrow to pull the cassette completely out e Hinged cover 1 Caution Turn the mains power supply switch off and detach the power cord before replacing input fuses 2 Open the fuse compartment by inserting a small flat blade screwdriver into the slot at the top of the power module Twist the screwdriver 8 turn to release the cover then pull out the hinged compartment which opens out 3 Insert the screwdriver above the arrow on one fuse cassette catch the cassette end and slowly slide it completely out of the module 4 Pull the fuse out of its cassette and inspect If the fuse element is burned or bro ken replace the fuse with an identical type If the fuse appears to be intact check it with a multi meter A reading of 1 W or less indicates the fuse is still usable 5 After p
8. are set in the edit mode Pressing the EDIT key toggles between the first editable screen and the protocol identification screen For each proto col the eleven different screens accessible in edit mode are Screen 1 rehydration time and temperature Screen 2 IEF run conditions temperature and maximum current strip Screens 3 11 the nine steps available for IEF conditions voltage change pattern voltage and step duration This section covers the programming editing or verifying of all parameter values 1 Select the protocol to be created or edited from the main screen Place the cursor under the protocol number field using the right and left arrow keys and then for ward to the desired protocol number using the up and down arrow keys Protocolc 41 NAME 2 Press the EDIT key to access the edit mode The first screen in edit mode contains fields for rehydration time and temperature Rehydration 00 00Hrs at 20 C To adjust the rehydration duration move the cursor using the right or left arrow key to the selected digit in the Hrs field Note that the cursor wraps around from left to right and right to left stopping only in the fields that can be edited Once the cursor is in place press the up or down arrow keys to adjust the value sl Rehydration 2000 d Hrs at The available time range is 0 99 59 h with 10 min resolution Hence only the first three digits are editable A
9. automatically to perform the separation The IPG strips are 3 mm wide and 0 5 mm thick after rehydration To rehydrate 1 Prepare the strip holder s Select the strip holder s corresponding to the IPG strip length chosen for the experiment 7 11 13 18 or 24 cm Wash each holder with strip holder cleaning solution supplied to remove residual protein Rinse thor oughly with double distilled water Use a cotton swab or a lint free tissue to dry the holder or allow it to air dry The holder must be completely dry before use Prepare the rehydration solution The Protocol Guide contains a commonly used recipe Required volumes per strip are itemized in the table below IPG strip length Total volume per strip holder cm ul including any sample 7 125 11 200 13 250 18 350 24 450 Pipet the appropriate volume of rehydration solu tion into each holder Deliver the solution slowly at a central point in the strip holder channel away from the sample application wells Remove any larger bubbles Important To ensure complete sample uptake do not apply excess rehydration solution Place the IPG strip Remove the protective cover from the IPG strip Position it with the gel side A down and the pointed anodic end of the strip directed toward the pointed end of the strip holder o Pointed end first lower the strip onto the solution To help coat the entire strip gently lift and lower the strip and slide it ba
10. cups can be placed almost anywhere along the length of the IPGphor strip holder that is not blocked by a pro trusion For proper sealing of the cup to the gel the feet of the sample cup must all rest on the bottom of the channel The cover is used to ensure that the electrodes stay in place and that they are in good contact with the IPG strip The cover also applies gentle pressure to ensure that the strip holder makes good contact with the IPGphor separation platform and power supply contact pads when the safety lid is closed 1 3 Important Information Instrument operation gt y y Caution IPGphor is a high voltage instrument that can cause fatal electrical shock if the safety features are disabled The safety lid must be properly latched before starting a protocol Exceeding the recommended current limit of 50 pA per IPG strip can cause the strip to burn and may damage the instrument During isoelectric focusing do not lean on the safety lid do not apply excess pressure or uneven weight to the lid and do not place any items on the lid Such pressure could cause arcing between the strip holder electrodes and the electrode areas damaging the instrument Plug the instrument into a properly grounded outlet Always disconnect the power cord before servicing Ensure that the cooling vents at the front back and bottom of the instrument are not obstructed Operate IPGphor on a level surface Avoid spilling liquids on the bo
11. or down arrow key to toggle between Hrs and Vhrs To set the digits first move the cursor to the desired digit location using the right and left arrow keys and then change the value using the up and down arrow keys The original factory programmed time for each step is 0 00 and should be set to 0 00 for all unused steps The Vhrs value is calculated according to the explanatory note at the left but these values can be set in the same manner as the Hrs values 1 Step n hold e t 1 Step n hold 500 v LAIT ell OR Programming example Voltage profile The set voltage is the volt age limit Under conditions of limiting current it may take some time for the maximum voltage to be reached and under certain power condi tions it may not be reached at all Simplified 4 step protocol and resulting voltage profile To illustrate the two voltage change patterns a simplified example shows program steps and the resulting voltage profile Note that step 4 ends the protocol For recommended IEF protocols see the IPGphor Protocol Guide 10000 8000 6000 Voltage V 4000 2000 Gradient Step n hold 1 Step n hold 500V 1 00 Hrs S 2 Gradient 2000V 1 00 Hrs 3 Step n hold 8000V 2 00 Hrs S 4 Step n hold OV 0 00 Hrs Step n hold a Pes pes H H P programmed voltage limit DU D aca actual voltag
12. p k 1 Prepare the sample in a solution similar in composition to the rehydration solution used 2 Pipet the sample into either or both of the lateral wells at either end of the strip holder Introduce the sample below the cover fluid Up to 7 5 pl of sample solution can be added to each side i e 15 pl per well or 30 ul max if both sides of both wells are used 17 18 3 5 Running a protocol 3 5 1 Final assembly 1 Place the strip holders The IPGphor platform has two electrode areas the larger area is the positive electrode anode and the smaller area is the negative electrode cathode Position the strip holder on the platform as shown below The pointed end of the holder is over the anode pointing to the back of the unit and the blunt end is over the cathode Guidemarks along the sides of the platform show approximate positioning for each strip holder size 7 11 13 18 and 24 cm Check that each of the two external electrode contacts makes metal to metal contact The pointed end of the strip holder is in the anodic electrode area External electrode contacts 2 underneath one in each electrode area Anodic electrode area Cathodic electrode area 2 Close the safety lid At least two of the three pressure pads under the safety lid must press gently against the cover of each strip holder to ensure contact between electrodes and electrode areas Important Pressure on the sa
13. thus the location of the acidic end of the immobilized gradient Embedded electrodes provide electrical connection between electrode areas and the gel Each of the two wells can accept up to 15 pl of sample 7 5 pl on either side of the strip in excess of the rehydration volume i e 30 ul maximum for both wells Introduce the sample through the wells if it is not included in the rehydration solution The cover protects the IPG strip during rehydration and the shape of the underside of the cover ensures electrical continuity during IEF by pressing strip against the electrode while allowing electrolysis gases to escape Rehydration and separation progress can be monitored visually through the clear cover The cup loading strip holders have two areas of metal plating on the bottom that extend up the sides of the strip holder These plated areas make contact with the power supply electrode areas when placed on the IPGphor separation platform Protrusions along the channel inside the strip holder align the rehydrated IPG strip keeping it straight and centered when placed inside the strip holder The anodic end of the strip holder is somewhat pointed to indicate the direction of placement of the pointed IPG strip The movable electrode clips can be placed anywhere along the strip holder where the electrode bosses make electrical contact with the conducting rails on the sides of the strip holder IPG strips from 7 cm to 24 cm long can be used Sample
14. 00 0 05 0 10 0 15 0 20 0 25 0 26 50V at 1pA 500V at 39A 500V at 15pA 500V at 10pA 500V at 10pA 500V at 9pA OVrhs 37Vhrs 78Vhrs 120Vhrs 162Vhrs 204Vhrs End of run XXX As isoelectric focusing proceeds the bromophenol blue tracking dye will migrate toward the anode and leave the IPG strip This generally occurs well before focusing is complete Note A colorless strip is no indication that IEF is complete If the tracking dye does not move then no current is flowing across the strip Check that the strip holder electrodes are in contact with the electrode areas See the Troubleshooting section for additional suggestions When the protocol ends audible beeps will sound and the following two screens will alternate Run completed STOP to reset The final instrument values are displayed Press the STOP key as indicated to reset the instrument 3 5 3 Pausing or stopping a protocol To pause or stop a protocol in progress press the STOP key Pause in step START to cont To continue the protocol press the START key To cancel the protocol press the STOP key again This will cause the instrument to respond as though the end of an uninter rupted protocol had been reached i e as described above the two final screens will alternate indicating Run completed press STOP to reset the instrument on one screen and indicating instrument final instrument values on the other 3
15. 02 47 80 6452 78 80 6429 60 80 6467 79 18 1004 40 IPGphor Isoelectric Focusing System First Dimension Protocol Worksheet Program steps Protocol _ Rehydration o Hrs at C IEF C __ UA max 1 Step n hold or Gradient V __ _ Hrs or Vhr S2 Step n hold or Gradient V __ __Hrs or Vhr S3 Step n hold or Gradient V cr Hrs or Vhr S4 Step n hold or Gradient V __ __Hrs or Vhr S5 Step n hold or Gradient Vo __ __Hrs or Vhr S Step n hold or Gradient V __ _ Hrs or Vhr S7 Step n hold or Gradient V __ _ Hrs or Vhr s8 Step n hold or Gradient V __ __Hrs or Vhr SO Step n hold or Gradient V __ __Hrs or Vhr 0 amersham pharmacia biotech IPG strip preparation Rehydration volume circle one IPG strip length cm 7 11 13 18 24 rehydration volume per strip pl 125 200 250 350 450 Rehydration solution Urea Detergent Reductant IPG buffer pH Dye Sample Water Total volume Notes Date Researcher Sample reagent final conc volume or weight IPGphor Isoelectric Focusing System First Dimension Protocol Worksheet Program steps Protocol _ Rehydration o Hrs at C IEF C __ UA max 1 Step n hold or Gradient V __ _ Hrs or Vhr S2 Step n hold or Gr
16. DIT key switches the display to edit mode in which protocol steps can be programmed See section 3 2 Pressing the EDIT key in edit mode switches the display back to the identification screen for the protocol being edited The left arrow key moves the cursor to the left Moving the cursor off the screen to the left repositions the cursor at the bottom right of the screen The right arrow key moves the cursor to the right Moving the cursor off the screen to the right repositions the cursor at the top left of the screen The up arrow key increases the value of the selected field or digit and the down arrow key decreases the value of the selected field or digit The START key begins the selected protocol The STOP key pauses the protocol Pressing the STOP key a second time cancels the protocol in progress The HV ON lamp indicates that the high voltage power supply is on and isoelectric focusing is in progress This lamp is not illuminated during the rehydration phase of a protocol Up to 12 fixed length strip holders of the same length or 9 variable length cup loading strip holders can be placed on the platform for rehydration and IEF The embedded electrode areas provide the electrical connection between strip holder electrodes and the integral power supply Platform temperature is controlled by Peltier thermoelectric modules Both electrode areas are gold plated copper Placing a strip holder so that it bridges both electrode area
17. IPGphor Isoelectric Focusing System User Manual d 80 6415 35 Rev A1 11 00 amersham pharmacia biotech 1 IPGphor Function and Description A EE 1 2 Instrument components lia ee Ga Sa Ne e A 1 3 Important information Lla vues i esa a dama ms deen 1 4 Specifications 2 Instrument Set Up Esc PIC the EIERE oc eis dera cet es titi use idees 2 2 Power connection LLeida als ed ee capes its Ne Mrs 2 3 Optional Serial port connector for a printer or computer connection 3 Operating Instructions Sle POWON UD oi e eu both ae Hi hw Sa m Sede PA 3 2 Creating and editing protocol programs 3 2 1 Protocol parameters 3 2 2 Editing protocol parameters 3 2 3 Naming a protocol 3 3 IPG strip rehydration and sample application IPGphor Cup Loading Strip Holder 3 4 IPG strip rehydration and sample application Fixed length IPGphor Strip Holder 3 5 RUNNIN A DESEN q onde ak das ds pes ESE que 3 5 1 Final assembly 3 5 2 Starting isoelectric focusing 3 5 3 Pausing or stopping a protocol 3 5 4 Printing a log of instrument conditions 4 Care and Maintenance 5 Troubleshooting 6 Customer Service Information 6 1 Technical service and repair 6 2 Ordering information ss n EE EN EE Proto
18. adas La garant a incluida en las condiciones de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entre gadas por Amersham Pharmacia Biotech Amersham Pharmacia Biotech no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de uti lizaci n y otras consecuencias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto Copyright O 2000 Amersham Pharmacia Biotech Reservados todos los derechos No est permitida la reproducci n ni el alma cenaje en un sistema de recuperaci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attenta mente l intero contenuto del presente manuale Italiano Il simbolo del fulmine all interno di un triangolo equilatero indica all utente la presenza di un rischio di esposizione ad alte tensioni Si prega di inviare eventuali commenti al presente manuale a Amersham Pharmacia Biotech Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Pharmacia Biotech si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilita Amersham Pharmac
19. adient V __ __Hrs or Vhr S3 Step n hold or Gradient V cr Hrs or Vhr S4 Step n hold or Gradient V __ __Hrs or Vhr S5 Step n hold or Gradient Vo __ __Hrs or Vhr S Step n hold or Gradient V __ _ Hrs or Vhr S7 Step n hold or Gradient V __ _ Hrs or Vhr s8 Step n hold or Gradient V __ __Hrs or Vhr SO Step n hold or Gradient V __ __Hrs or Vhr 0 amersham pharmacia biotech IPG strip preparation Rehydration volume circle one IPG strip length cm 7 11 13 18 24 rehydration volume per strip pl 125 200 250 350 450 Rehydration solution Urea Detergent Reductant IPG buffer pH Dye Sample Water Total volume Notes Date Researcher Sample reagent final conc volume or weight Amersham Pharmacia Biotech UR Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany www apbiotech com Hoefer Immobiline IPGphor and PlusOne are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia amp Upjohn Inc Triton is a trademark of Union Chemicals and Plastics Co All goods and services are sold subject to
20. c program will stop and a message will indicate the source of the fault note the fault and press the START key to skip to the next test If any failure is detected call the technical service department Once the diagnostic program is successfully completed the main screen indicates that the instrument is ready as shown below The main screen is Protocol 1 Name The default location of the cursor is always under the protocol number 1 Select any protocol by pressing the up and down arrow keys to display the number of the desired protocol IPGphor The START key Protocol ll begins the selected protocol The left arrow key The EDIT key switches the dis The right arrow key moves the cursor to the play to edit mode in which pro moves the cursor to left Moving the cursor tocol steps can be programmed the right Moving the off the screen to the left Pressing the EDIT key in edit cursor off the screen repositions the cursor mode switches the display back to the right repositions at the bottom right of to the identification screen for the the cursor at the top the screen protocol being edited left of the screen 3 2 Creating and editing protocol programs 3 2 1 Protocol parameters The IPGphor can store up to ten protocols Programmable protocol parameters include duration and temperature of the rehydration phase and duration tempera ture and power parameters for up to nine IEF steps for each protocol
21. ck and forth along the sur face of the solution tilting the strip holder slightly 4 as needed to assure complete and even vvetting Finally lower the cathodic square end of the strip into the channel making sure that the IPG gel con tacts the strip holder electrodes at each end The ee gel can be visually identified once the rehydration ii d a solution begins to dye the gel Be careful not to trap bubbles under the strip Note The IPG strip backing is impermeable do not apply the sample to the back of the strip 5 Apply Immobiline Drystrip Cover Fluid to minimize evaporation and urea crystal lization Pipet the fluid dropwise into one end of the strip holder until one half of the strip is covered Then pipet the fluid dropwise into the other end of the strip holder adding fluid until the entire IPG strip is covered 6 Place the cover on the holder Pressure blocks on the underside of the cover assure that the strip maintains good contact with the electrodes as the gel rehydrates 7 Rehydration can proceed on the bench top or on the IPGphor unit platform Ensure that the holder is on a level surface A minimum of 10 h is required for rehydration overnight is recommended Alternatively the rehydration period can be programmed as the first step of an IPGphor protocol This is especially conve nient if temperature control during rehydration is a concern or if a low voltage is applied during rehydration Optional Sampl
22. col Worksheets _ 000 Renseignements importants d utilization Pour une bonne compr hension et une utilisation en s curit maximale il convient de lire enti rement ce manuel Fran ais Le symbole de l clair dans un triangle quilat ral a pour objet d attirer l attention de Putilisateur sur un danger d exposition la haute tension Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser a Amersham Pharmacia Biotech Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Pharmacia Biotech se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Amersham Pharmacia Biotech garantit l utilisateur que le produit livr a subi avec succ s tous les essais pr vus pour s assurer qu il est conforme aux sp cifi cations et normes en vigueur La garantie incluse dans les conditions de livrai son n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Amersham Pharmacia Biotech La soci t Amersham Pharmacia Biotech ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisation incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commerciales tout emp chement d utilisation et tout autre
23. der Conventionally IPG strips are rehydrated in one step and loaded and run in a second step In the IPGphor system this is done by first rehydrating the gels in the Immobiline DryStrip Reswelling Tray then transferring the gels to a strip holder for separation The strip holder may be either fixed length IPGphor Strip Holder or a variable length IPGphor Cup Loading Strip Holder The IPGphor Cup Loading Strip Holder is suitable for first dimension isoelectric focusing IEF of proteins on immobilized pH gradient IPG gel strips 3 mm wide from 7 cm to 24 cm long IEF is performed with the gel side facing up Samples are applied in a localized region through an open bottom loading cup Cup loading has been found to improve protein focusing patterns particularly on basic IPG strips pH 6 9 and 6 11 Under conditions where substantial water trans port electroendosmosis accompanies focusing such as with protein loads in excess of 1 mg the faceup mode frequently yields better resolution Detailed instructions for the use of the Cup Loading Strip Holder are found in the IPGphor Cup Loading Strip Holder User Manual Electrode clip Sample cup 15 Important Handle the holders with care as they are brittle and fragile 16 3 4 IPG strip rehydration and sample application Fixed length IPGphor Strip Holders IPGphor fixed length strip holders allow IPG strips to be rehydrated and samples loaded in one step before proceeding
24. dy of the instrument Avoid scratching the electrode areas IPG strips strip holders and reagents m The strip holders are very brittle and fragile Handle carefully Do not bump or drop Always wear gloves when handling IPG strips and the equipment that comes in contact with them This will help minimize protein contamination which can result in artifactual spots in the silver stained 2 D patterns Clean strip holders with the strip holder cleaning solution provided Clean all other components that come in contact with the IPG strip or the sample with a detergent designed for glassware Rinse well with distilled water Use the appropriate rehydration volume for the IPG strip length Solutions containing urea may be warmed briefly Do not heat any solutions containing urea above 30 40 C as isocyanate a urea degradation product will carbamylate the proteins in the sample thus changing their isoelectric points All chemicals should be of the highest purity Double distilled water should be used Informations importantes Pr cautions d emploi gt y y Attention l IPGphor est un instrument tr s haut voltage pouvant causer un choc lectrique mortel si les conditions de s curit ne sont pas respect es Le couvercle de s curit doit tre convenablement verrouill avant de d marrer un protocole D passer les limites de courant recommand es 50 uA un bande IPG peut amener les bandes br ler et end
25. e 2 3 4 Time hours Note When programmed in Vhrs mode the IPGphor may skip to the next step following the final programmed step Occasionally low actual voltages due to excessive current causes protocol steps to fall short of the programmed Vhrs for that step The software compensates by skipping to the next step to complete the total Vhrs that were programmed into the protocol 5 To verify the protocol scroll through each step place the cursor in the step num ber field using the right and left arrow keys and then advance through the steps using the up and down arrow keys Edit as required by placing the cursor with the right and left arrow keys and adjusting the value with the up and down arrow keys The Protocol Worksheets at the back of the manual can be useful in this step Make sure that the voltage field in all unused steps displays a value of 0 and that the value in the Hrs field is 00 00 Note Pressing the up arrow key in screen 9 wraps back to the rehydration screen Press the EDIT key to save any changes and exit the edit mode The protocol identification screen will appear 13 14 3 2 3 Naming a protocol Protocols are identifiable by their number 1 10 and by sixteen alpha numeric charac ters in the bottom row of the protocol identification screen All 26 letters of the Roman alphabet a blank space and the digits 0 9 are available for each character space Protocol names are programmed from the
26. e rehydration under voltage If desired the sample can be absorbed by the IPG strip under low voltage This may improve the uptake of the high molecular weight proteins To perform rehydration under voltage set the rehydration time to 0 00 and program Step 1 of the protocol for low voltage 30 100 V for 10 12 h Program additional steps as desired to achieve complete focusing Optional Apply electrode pads just prior to IEF Under certain conditions such as prolonged focusing water may migrate toward one end of the strip causing the other end to begin drying out This effect can be mini mized by placing paper electrode pads between the IPG strip and each strip holder electrode just before IEF Electrode pads may also absorb ions that would otherwise accumulate at the ends of the IPG strip and possibly interfere with the separation 1 To apply cut two 3 mm wide electrode pads from a paper IEF electrode strip 18 1004 40 Place on a clean flat surface such as a glass plate and soak with deionized water Remove excess water by blotting with tissue paper Important Electrode pads must be damp not wet 2 Using forceps or tweezers lift one end of the rehydrated IPG strip Position an electrode pad over the electrode then lower the strip back in place Repeat at the other end Optional Apply sample after rehydration If the sample was not applied by inclusion in the rehydration Wd solution it can be applied immediately prior to IEF
27. ell as all parameters will appear S1 20 40 mA OW 1000V 6 40Hrs The actual platform temperature is indicated To change the value use the right and left arrovv Reys to place the cursor in the temperature field and use the up and down arrow keys to adjust the value Do not press the EDIT key to change this value The average current per IPG strip pA the total instantaneous power delivered W and actual voltage V are indicated If the current is the limiting parameter the A in nA will flash The total elapsed time since the IEF protocol started excluding rehydration or alter natively the voltage hours Vhr delivered is indicated in the lower right To access the Vhr value move the cursor to the Hrs field with the right and left arrow keys and toggle between Hrs and Vhr with the up and down arrow keys To change the hours remaining while the protocol is in progress press the EDIT key to display the time remaining 06 40Hrs 00 59Hrs Total Step Move the cursor under the digit you wish to set using the right and left arrow keys and adjust the value using the up and down arrow keys To stop the step in progress change the Step values to 00 00 Hrs After a few seconds the status screen will appear automatically to show the step in progress Amersham Pharmacia Biotech IPGphor Protocol No of strips 1 IEF Params 50 pA strip at 20 C S1 Step n hold 500V 3 00Hrs 0
28. f steps and total hours excluding rehydration time Starting prot _ _steps __ __Hrs If a rehydration period was programmed the rehydration status screen will indicate the number of hours elapsed and the actual platform temperature Rehydration 00 01Hrs at 20 C 19 The voltage change pattern is indicated by the follow ing symbols step n hold gradient Note The indicated wattage is the total power delivered to all strips and will often read 0 VV because any value less than 0 5 is rounded down to O If the delivered power exceeds 0 4 W per strip stop the run and reduce the V or pA set tings 20 To change rehydration duration press the EDIT key to display the time remaining Remaining rehydr 09 00Hrs Move the cursor under the digit you wish to set and change it using the up and down arrow keys To end this step set all digits to 0 This screen is automatically replaced by the regular status screen after a few seconds The temperature field can be changed directly without pressing the EDIT key use the right and left arrow keys to place the cursor in the temperature field and use the up and down arrow keys to adjust the value It may take up to 30 minutes to reach the set temperature depending on ambient conditions Isoelectric focusing will begin after the rehydration step is complete The HV ON lamp will light and the status screen showing the step currently in progress as w
29. fety lid could cause arcing between the strip holder electrodes and the electrode areas and may damage the instrument 3 5 2 Starting isoelectric focusing 1 Advance to the protocol identification screen by placing the cursor in the protocol number field using the right and left arrow keys and selecting the protocol num ber using the up and down arrow keys x Protocol H1 NAME 2 Press the START key The first screen requests that you enter the number of IPG strips in place The IPGphor accommodates up to twelve fixed length strip holders of the same length or nine cup loading strip holders To enter the number use the right and left arrow keys to position the cursor and the up and down arrow keys to select the number 1 12 Number of strips If the safety lid is not properly latched the following message will indicate the required action Lid open step 1 close to continue After securing the safety lid press the START key again Important During IEF mb do not lean on the safety lid m do not apply excess pressure or uneven weight to the lid and m do not place any items on the lid Once the protocol begins each step is carried out automatically and instrument status for each step is indicated by the screen Platform temperature and time remaining can be changed while the protocol is in progress and each such possibility is discussed in turn below The first screen will indicate the protocol number o
30. g there Refer to the Troubleshooting section of the IPGphor Protocol Guide 24 Customer Service Information 6 1 Technical service and repair Amersham Pharmacia Biotech offers complete technical support for all 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 Amersham Pharmacia Biotech representative Important Request a copy of the Amersham Pharmacia Biotech Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed 6 2 Ordering information IPGphor isoelectric focusing unit Qty Code No Basic unit Order strip holders separately 1 80 6414 02 Protocol Guide IPGphor 1 80 6415 73 2 D Electrophoresis brochure for complete information on sample 1 18 1124 82 preparation and rehydration chemicals systems for first and second dimension blotting staining and evaluation Fuses 1 6 A 250V 5x20mm 5 80 6108 69 IPGphor Strip Holder Covers 7 cm 2 80 6455 44 11 cm 2 80 6455 63 13 cm 2 80 6455 82 18 cm 2 80 6456 01 24 cm 2 80 6479 76 Strip holders for use vvith Immobiline DryStrip gels 7 cm complete 6 80 6416 11 11 cm complete 6 80 6416 30 13 cm complete 6 80 6416 49 18 cm complete 6 80 6416 68 24 cm complete 6 80 6469 88 7 cm complete 1 80 6416 87 11 cm complete 1 80 6417 06 13 cm complete 1 80 6417 25 18 cm complete 1 80 6417 44
31. hout prior notice Warranty and Liability Amersham Pharmacia Biotech guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the product has been installed and used according to the instructions supplied by Amersham Pharmacia Biotech Amersham Pharmacia Biotech shall in no event be liable for incidental or con sequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Copyright 2000 Amersham Pharmacia Biotech All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company IPGphor Isoelectric Focusing System Function and Description The IPGphor Isoelectric Focusing System performs first dimension isoelectric focusing IEF in 2 dimensional 2 D electrophoresis The system comprises Immobiline DryStrip gel strips which contain an immobilized pH gradient IPG strip holders which serve as both rehydration and IEF chambers for the IPG strips and the IPGphor unit which includes a high voltage DC power supply solid state temperature control and can be programmed with up to ten user defined IEF protocols Program
32. ia Biotech garantisce che prima della consegna il prodotto stato collaudato a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato installato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Pharmacia Biotech Amersham Pharmacia Biotech non potra essere ritenuta responsabile di inciden ti o danni consequenziali inclusi ma non limitati a perdite di profitti mancato guadagno perdite di affari difetti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto Copyright 2000 Amersham Pharmacia Biotech Tutti i diritti riservati Nessuna parte della presente pubblicazione pu essere riprodotta conservata in sistemi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore Please read this entire manual to fully understand the safe and effective use of this product Important user information S English The lightning symbol vvithin an equilateral triangle is intended to alert the user to the risk of exposure to high voltages Should you have any comments on this manual we will be pleased to receive them via email at ts ep am apbiotech com or at Amersham Pharmacia Biotech Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Pharmacia Biotech reserves the right to make changes in the specifi cations wit
33. ip The original factory programmed value is 50 pA per IPG strip This is the maxi mum recommended setting to prevent overheating and possible damage to the strip or the unit To adjust the current limit use the left and right arrow keys to place the cursor under one of the three active pA digits and use the up and down arrow keys to select the value within the range 0 200 pA per strip Note The total deliverable amperage is 1500 pA IEF parameters 21 C GOJA strip To advance to the next edit screen move the cursor to the IEF field and press the up arrow key The third screen encountered in edit mode is the first in a series of nine program mable IEF steps in which the voltage change pattern voltage and step duration are set Each step is identified by S and the step number and can be accessed by placing the cursor under the step number using the right and left arrow keys To advance to the next step press the up arrow key and to return to a previous step press the down arrow key 11 Note When toggling from Hrs to Vhr the value in the Vhrs field is automatically calculat ed according to the selected voltage change pattern For Step n hold Vhr is the product of hours and voltage For Gradient Vhr is the product of hours and the mean of the voltage limit set for the current step and the previous step Absolute Vhr limits can be set to any value from 1 99999 Vhr 12
34. lacing a good fuse into the cassette slide it into the power module making sure the arrow on the cassette points to the right in the same direction as the guide arrows on the inside of the compartment door 6 Repeat steps 3 to 5 for second cassette 7 Close the fuse compartment cover and gently press it into the power module until it snaps shut 8 Plug the power cord into the unit and turn the mains power switch on 23 Troubleshootin i Problems indicated by LCD messages Lid open step 1 close to continue The safety lid is not properly closed When the safety lid is open the system has an automatic voltage cutoff safety feature In order for the protocol to proceed the safety lid must be closed Locked screen in edit mode Turn off the mains power switch to reset the instrument Blank display If no electrical components are functioning e g HV lamp does not light and the cooling fans are motionless check the fuses in the mains power module Diagnostic program indicates component failure Sr Note the component that failed and press the START key to continue through the diagnostic program Call your local Amersham Pharmacia Biotech sales office for further information on how to remedy the failure Power delivery Current too low or zero At least two of three pressure pads under the safety lid should press gently against the strip holders to ensure electrical continuity between the strip hold er elect
35. mable functions include rehydration time platform temperature maximum current limit voltage change pattern voltage for each step in the isoelectric focusing program and step duration IPG strips are available in five lengths 7 11 13 18 and 24 cm and a number of pH ranges several linear and nonlinear listed in section 7 2 Ordering information Two types of strip holders are available a fixed length strip holder and a variable length cup loading strip holder Once the IPG strip is in place in a fixed length strip holder no fur ther handling is required sample is introduced either during or after the rehydration step and the holder is placed on the IPGphor electrode platform for isoelectric focusing Up to 12 strip holders of the same size can be accommodated for any one protocol Cup loading strip holders accommodate 3 mm wide strips from 7 to 24 cm long After rehydration strips are placed in the strip holder and the sample cup positioned as desired for loading in a particular zone Up to 9 cup loading strip holders can be accom modated at one time 1 1 Unpacking Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local Amersham Pharmacia Biotech 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
36. minimum of 10 h is required for the rehydration of IPG strip gels but overnight is recommended This protocol step can also be used as a simple timer to start the protocol at a convenient time If the IPG strips are already rehydrated and ready for immediate focusing set the rehydration duration to 00 00 Hrs Note Maintaining the actual platform temperature at lt 18 C or gt 25 C may require placement of the IPGphor unit in a location of lower or higher than ambient temperature e g a cold room Screen 2 IEF parameters Screens 3 11 IEF protocol steps To adjust the rehydration temperature use the left and right arrow keys to place the cursor and the up and down arrow keys to select the value within the range of 15 30 C The original factory programmed value is 20 C Rehydration 10 00Hrs at DA To advance to the next edit screen move the cursor to the Rehydration field and press the up arrow key Rehydration 10 00Hrs at 22 C The second screen in edit mode contains fields to set the platform temperature and current limit for all isoelectric focusing steps IEF parameters 20 C 50pA strip The original factory programmed IEF temperature is 20 C To adjust the temper ature use the left and right arrow keys to place the cursor in the active C field and use the up and down arrow keys to select the value within the range of 15 30 C IEF parameters Tere 5O0pA str
37. ommager l instrument Durant Piso lectrofocalisation ne pas s appuyer sur le couvercle de s curit ne pas exercer de pression excessive ni de poids inutile sur le couvercle et ne pas placer d objets sur le couvercle Une telle pression pourrait provoquer un arc lec trique entre les lectrodes des cassettes et la plate forme de migration porte lec trodes ab mant l instrument Brancher l instrument une prise de terre appropri e Toujours d connecter le cordon d alimentation avant la maintenance S assurer que les ventilations devant et l arri re de l appareil ne sont pas obstru es Utiliser PIPGphor sur une surface plane Eviter de renverser des liquides sur l instrument Eviter de rayer la plate forme de migration porte lectrodes M ches IPG cassettes et r actifs Les cassettes sont tr s fragiles Manipulez les avec pr caution vitez de les heurter ou de les laisser tomber Toujours mettre des gants pour manipuler les bandes IPG ainsi que I quipement en contact avec celles ci Ceci aide minimiser la contamination prot ique qui peut occasionner des spots artefactuels sur le gel 2D apr s la coloration l argent Nettoyer les sarcophages avec la solution d di e a cet effet Nettoyez tous les composants qui rentrent en contact avec les bandes IPG ou l chantillon avec un d tergent pour mat riel en verre Bien rincer avec de l eau distill e Utilisez le volume ap
38. propri de r hydratation Les solutions contenant de l ur e peuvent tre chauff es bri vement Ne pas chauffer de solution contenant de l ur e plus de 30 40 C car l isocyanate un produit de d gradation de l ur e produira une carbamylation des prot ines de chantillon changeant ainsi leurs points iso lectriques Tous les produits chimiques doivent tre de la plus grande puret Veillez utiliser de l eau doublement distill e 1 4 Specifications Working surface Electrode areas Strip holder capacity Platform temperature Strip holders Material Holders for IPG strip lengths User interface Control panel Programmable parameters Protocol capacity Serial port Operating environment Temperature Relative humidity Power requirements Line voltage Frequency Power consumption Power outputs Voltage Current Power Physical parameters Dimensions h x w x d Weight Safety Safety features Safety certifications Gold plated copper Up to 12 fixed length up to 9 cup loading 18 25 C 1 C Aluminum oxide ceramic base acrylic cover polycarbonate platinum on titanium 7 11 13 18 and 24 cm 7 key membrane keypad Liquid crystal display LCD 2 lines 16 characters per line Rehydration time platform temperature maximum current limit per strip step voltage step voltage change pattern and step duration 10 with up to 9 steps each RS232C 1200 baud 15 35
39. protocol identification screen not in edit mode 1 First advance to the protocol to be named Place the cursor under the protocol number using the right and left arrow keys and then advance to the desired pro tocol number using the up and down arrow keys Protocol 8 2 To change any letter move the cursor to that location using the right and left arrow keys and then scroll through the 37 available options using the up and down arrow keys Once the desired letter appears move the cursor to the next character to be set The original label NAME occupies 4 of the 16 available characters To change the label to a more descriptive name such as 11 CM 3 101 requires scrolling through the alpha numeric options for each of the 11 digits in the name Note Be sure to count the total number of characters required including blank spaces before beginning Abbreviate as required to fit within the allotted 16 character spaces For this example begin by placing the cursor under the third or fourth depending on your preference character space using the right and left arrow keys and then press the up arrow key until 1 appears Protocol 1 Continue setting a character for each required space until the protocol name is spelled out Protocol 1 11 CM 3 10L Components of the IPGphor Cup Loading Strip Holder 3 3 IPG strip rehydration and sample application IPGphor Cup Loading Strip Hol
40. r connection is at the left of the unit near the bottom The type of cable required straight serial or null modem depends on the type of device DTE or DCE that it is connected to If the printer or computer is configured to receive data on pin 2 and transmit on pin 3 a regular straight serial cable is required For any other configuration a null modem cable is required Pin number assignments and device set up details follow IPGphor RS232C signal and pin number assignments Pin 2 Transmit Pin 3 Receive Pin 5 Ground Other pins Not connected The IPGphor requires these settings in the device receiving data Baud rate 1200 Data bits 8 Stop bit 1 Start bit 1 Parity None Flovv control None IPGphor control panel The STOP key pauses the protocol Pressing the STOP key a second time cancels the protocol in progress and pressing it a third time resets the instrument to start another protocol The up arrow key increases the value of the selected field or digit and the down arrow key decreases the value of the selected field or digit Operating Instructions The following sections will guide you through each stage of the isoelectric focusing process using the IPGphor 3 1 Power up Turn on the mains power switch I located on the right side of the rear panel This acti vates a self diagnostic program that runs for approximately 10 seconds A series of screens indicates progress If any component fails the diagnosti
41. risques ayant un rapport avec l utilisation du produit mais sans aucune limitation quant a la nature de ces dommages Copyright 2000 Amersham Pharmacia Biotech Tous droits r serv s La reproduction le stockage dans un syst me de r cup ration d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou en partie sont strictement interdits sans autorisation pr alable crite de la soci t Wichtige Benutzerinformationen F r ein vollst ndiges Verstandnis und eine sichere Handhabung dieses Produktes ist es notwendig daf der Benutzer dieses Handbuch vollst ndig durchliest Deutsch Ein Blitzsymbol in einem gleichseitigen Dreieck soll den Benutzer auf die Gefahr anliegender Hochspannungen hinweisen Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Amersham Pharmacia Biotech Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Pharmacia Biotech beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and Haftung Amersham Pharmacia Biotech garantiert da das gelieferte Produkt sorgf ltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem f den von Amersham Pharmacia Biotech geliefer ten Anwei
42. rodes and the electrode areas on the platform Ba The gel must be evenly and completely rehydrated to conduct current Make sure the proper amount of rehydration solution is applied to the IPG strip holder and allow a minimum of 10 hours for rehydration Refer to the Troubleshooting section of the IPGphor Protocol Guide Voltage limit not reached The ionic strength of the rehydration solution is too high reduce the IPG buffer concentration use a mixed bed ion exchange resin to remove ionic breakdown products of urea of other additives Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM Refer to the Troubleshooting section of the IPGphor Protocol Guide Sparks or burning in strips Sr Reduce the current limit Do not exceed 50 pA per strip Sr Prevent the IPG strip from drying out by always applying Immobiline DryStrip Cover Fluid during rehydration Ensure that the IPG strip is fully rehydrated along the entire length of the strip The IPG strip should be in complete contact with the correct volume of rehydration solution Remove any air bubbles trapped under the IPG strip Desalt the sample or prepare the sample so thaat the salt concentration is less than 10 mM De ionize additives to the rehydration solution Excessive charged material in the sample or rehydration buffer lead to electroendomosis which will dry out the middle of the strip possibly leading to arcing and burnin
43. s completes the electrical circuit once the safety lid is in place Each holder must be oriented properly Negative cathode area Place the flat end of the holder in the shorter electrode area visually verifying that there is metal to metal contact between the electrode on the bottom of the holder and the electrode area Positive anode area The pointed end of the holder points toward the back of the unit and the electrode must rest on the longer electrode area the position of the tip should correspond to the guidemarks on the edge of the platform 7 11 13 18 and 24 cm The safety lid must be properly latched before power can be applied The pressure pads in the lid secure the strip holders A minimum of two pads should contact the cover of each strip holder to ensure an electrical connection The strip holders are made of aluminum oxide ceramic of minimal thickness for efficient heat transfer This material is very brittle so the holders are fragile and must be handled with care The ceramic is treated with a special coating to minimize protein binding and must be cleaned only with the IPGphor Cleaning Solution supplied to retain that coating Each fixed length strip holder base holds a single IPG strip throughout rehydration and isoelectric focusing A match ing length holder is available for each size strip The holders like IPG strips have a flat and a pointed and The pointed end point is positive designates the anode and
44. sungen installiert und benutzt wurde Amersham Pharmacia Biotech bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbufen Einkommensverluste entgangene Gesch ftsabschl sse Verlust der Gebrauchsf higkeit oder andere Verluste die wie auch immer durch eine fehler hafte oder unsachgem e Verwendung des Produkts verursacht wurden Copyright 2000 Amersham Pharmacia Biotech Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Genehmigung durch das Unternehmen vervielf ltigt in einem Abrufsystem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Informaci n importante para el usuario Es Para comprender el producto y utilizarlo con seguridad Espanol es necesario leer este manual en su totalidad El s mbolo del rayo en un tri ngulo equil tero alerta al usuario sobre el riesgo de exposici n a altas tensiones Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Pharmacia Biotech Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Pharmacia Biotech se reserva el derecho a modificar las especifica ciones sin previo aviso Garant a y responsabilidad Amersham Pharmacia Biotech garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especificaciones publi c
45. ted parts Never use ketones hot strong acids or hydrocarbon solvents IPGphor Strip Holders are made from a thermally conductive aluminum oxide ceramic They have been surface treated to minimize adsorption and interaction with proteins The surface coating is mechanically robust and can withstand temperatures of up to 300 C however it can be damaged by prolonged exposure to extremes of pH IPGphor Strip Holder Cleaning Solution has been specifically formulated for removing pro tein deposits It will not damage the strip holder IPGphor Strip Holder Cleaning Solution 80 6452 78 can be ordered in 950 ml bottles from Amersham Pharmacia Biotech Cleaning procedure Clean strip holders after each first dimension IEF run Do not let solutions dry in the strip holder Cleaning may be more effective if the strip holders are first soaked a few hours to overnight in a solution of 2 5 IPGphor Strip Holder Cleaning Solution in water Strip holders may also be immersed in boiling 1 SDS 1 First rinse off the strip holder Use a mild liquid soap to remove any residual IPG cover fluid 2 Squeeze few drops of IPGphor Strip Holder Cleaning Solution into the strip holder slot Use a toothbrush and vigorous agitation to clean the strip holder 3 Rinse well with distilled or deionized water Thoroughly air dry the strip holders or dry well with a lint free tissue prior to use Wipe the platform and plate electrodes with a lab tissue or paper towel
46. the terms and condi tions of sale of the company within the Amersham Pharmacia Biotech group that supplies them A copy of these terms and conditions is available on request 2000 Amersham Pharmacia Biotech Inc All rights reserved Printed in the USA 0 amersham pharmacia biotech
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