Ettan IPGphor Cup Loading Strip Holder
Contents
1. Important Request a copy of the Amersham Biosciences Health and Safety Declara tion form before returning the item No items can be accepted for servicing or return unless this form is properly completed Care and cleaning Ceramic strip holders Gently clean the strip holders after each use Wash the holder with the Ettan IPGphor Strip Holder Cleaner Rinse thoroughly with water and then deionized water and allow the strip holders to air dry Do not use abrasive cleaning agents or pads as they may damage the metal conducting rails Take care to avoid chipping the strip holders Electrodes Rinse well with deionized water after use and allow to air dry Covers Wash with standard lab detergents rinse and air dry Customer service information Technical service and repair Amersham Biosciences offers complete technical support for all our products If you have any ques tions about how to use this product or would like to arrange to repair it please call or fax your local Amersham Biosciences representative p13 Ordering information Replacement parts code no Ettan IPGphor Cup Loading Strip Holder 3 pk complete 80 6459 43 Sample cups pack of 50 80 6459 81 Electrodes pack of 6 80 6464 94 Cover Cup Loading Strip Holder 80 6465 13 Ceramic Base Cup Loading Strip Holder 80 6459 62 without cover electrode and cups Accessory products quantity code no Immobi2. 6 11 1 2 5 h t 8500 VI 3 cm 3 10 L 3 10 NL 4 7 1 54 h 13000 V 3 cm 6 11 1 53 h 11600 V 8 cm 3 10 L 3 10 NL 4 7 3 6 h t 24600 Vh 8 cm 6 11 2 5 5 h 20600 Vh 8 cm 6 9 5 11 h 41600 Vh 8 cm 3 5 4 5 4 5 4 5 5 5 5 6 5 5 6 7 7 14h t 56600 Vh 24 cm 3 10 L 3 10 NL 4 7 L 3 7 NL 6 13 h 51600 Vh 24 cm 3 5 4 5 4 5 4 5 5 5 5 6 5 5 6 7 12 20 h 95600 Vh 24 cm 6 9 7 14 h 56 500 Vi The sample entry phase should be extended for high protein loads or for convenience if the strips are to be run overnight tTimes are estimates for completion of the volt hours required for focusing Depending on the conductivity of the rehydra tion solution and the length and pH range of the IPG strips the focusing run may not reach the final desired voltages It is more important that IPG s rips are focused for the required volt hour for complete focusing rather than achieving 8000 V Programming the IPGphor for the desired volt hour in the final phase will help ensure focusing Using preparative protein loads and high salt concentrations in the cup loading strip holder may cause cover oil to redistribute and flow out of the stripholder See Table 1 below Table 1 Troubleshooting To counteract oil leakage from Ettan IPGphor Cup Loading Strip Holder at high salt concentrations use the following running conditions current limit 50 yA per strip step duration step duration voltage voltage h min Vh gradient type
3. es notwendig da der Benutzer dieses Handbuch vollst ndig durchliest Ein Ausrufezeichen in einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und Wartungsanweisungen in der dem Ger t beiliegenden Dokumentation hinweisen Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and haftung Amersham Biosciences garantiert da das gelieferte Produkt sorgf ltig auf die Einhaltung der ver f fentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleis tungsanspr che gelten nur dann wenn das Produkt gem den von Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde Amersham Biosciences bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbu en Einkommensver luste entgangene Gesch ftsabschl sse Verlust der Gebrauchsf higkeit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden 2002 Amersham Biosciences Corp Alle Rechte vorbehalten Die vorliegende Ver ffentlichung darf nur mit vorhergehender schriftlicher Genehmigung durch das Unternehmen verviel
4. 1 300 3 00 900 Step n hold 3 1000 6 00 3900 Gradient 4 8000 3 00 13500 Gradient 5 8000 i Step n hold Total step duration and Vh as recommended for individual strip lengths p11 Troubleshooting Sample application at the anodic or cathodic end of the IPG strip usually gives the best results but the placement must be determined empirically for each sample type As a general rule anodic application is more successful on basic IPG strips problem possible cause remedy No electrical contact Plated ends of strip holder are not in proper contact with the Ettan IPGphor unit Electrode contacts are not placed correctly Check that the anode and cathode ends of the strip holder are in proper contact with the electrode contact areas on the Ettan IPGphor unit Check that the bosses on the inside of the electrode contacts are in contact with the plated side rails Sample leaks from cup Incorrect strip alignment Incorrect cup placement Check that the foot of the cup is not resting on a protrusion on the inside of the strip holder Check that the strip is centered inside the channel Cover fluid wicks out Excess cover fluid of the strip holder High salt in sample Do not add more than the recommended volume of cover fluid to cover the strip Excess oil may capillary wick out of the holder resulting in strips drying out during the run Use slower ramping up of voltage see Table 1 S
5. aci n y manten imiento del aparato Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el derecho a modificar las especificaciones sin previo aviso Garant a y responsabilidad Amersham Biosciences garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las especificaciones publicadas 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 entregadas por Amersham Biosciences Amersham Biosciences 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 utilizaci n y otras consecuen cias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto O 2002 Amersham Biosciences Corp Reservados todos los derechos No est permitida la reproducci n ni el almacenaje 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 Deutsch A Wichtige benutzerinformationen Fur ein vollst ndiges Verstandnis und eine sichere Handhabung dieses Produktes ist
6. ati 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 Fig 1 Components of the Ettan IPGphor Cup Loading Strip Holder system Ettan IPGphor Cup Loading Strip Holder The Ettan IPGphor Cup Loading Strip Holder is suitable for first dimension isoelectric focusing IEF of proteins on Immobiline DryStrip immobilized pH gradient IPG gel strips 3 mm wide from 7 cm to 24 cm long in the Amer sham Biosciences Ettan IPGphor Electrophoresis unit IEF is performed with the gel side facing up Samples are applied in a localized region through an open bottom loading cup Cup load ing has been found to improve protein focusing patterns particularly on basic IPG strips pH 6 9 and 6 11 Under conditions where substan tial water transport electroendosmosis accom panies focusing such as with protein loads in excess of 1 mg the faceup mode frequently yields better resolution Strip holder protrusions i D Cover lay I 27 plated rails Sample cup Electrode clips cup opening top electrode boss cup opening bottom feet standoff gel contact face um 80 6465 70 p2 Strip holder The strip holders are made of aluminum oxide ceramic for efficient heat transfer and tempera ture control durin
7. e as possible Fig 5 Side protrusions prevent feet from seating properly at certain points um 80 6465 70 p8 O Place the sample cup over the IPG strip Do not place the cup with the feet over an alignment protrusion The cup will not seat squarely and seal on the IPG strip The sample cup can straddle these protrusions however Press the cup down until fully seated against the bottom of the strip holder The feet on the sample cup stop the cup at a height above the floor of the strip holder sufficient to seal against the strip without cutting into it Fig 5 Make sure the cup and elec trodes are positioned properly prior to loading the sample in the cup N VE blocked loading cup foot area Fig 6 Pipette sample into the sample cup To confirm that the sample cup has sealed pipette a small amount of rehydration buffer without sample into the cup Fig 6 Check the seal Remove the solu tion prior to loading the sample Common causes of leaking include e A foot of the cup is resting on top of one of the protrusions Remove the cup and gently remove any excess solution from the top of the gel strip Reposi tion the cup and recheck for leakage e The gel strip is not centered in the channel Pipette up excess liquid from the strip holder being careful not to damage the IPG strip Remove the cup center the IPG strip reposition the cup and check again e The strip may not ha
8. es good contact with the IPGphor separation plat form and power supply contact pads when the safety lid is closed Required but not included Immobiline DryStrip Cover Fluid The Immobiline DryStrip Cover Fluid is required to ensure that the rehydrated Immobi line DryStrip IPG gels do not dry out during electrophoresis Without cover fluid the strips will dry out urea will crystallize and the sample will not focus properly Immobiline DryStrip Reswelling Tray The Immobiline DryStrip Reswelling Tray is required for proper strip rehydration The channel in the Cup Loading Strip Holder is too wide for proper absorption of the required volumes of rehydration solution Two trays are available One for 7 18 cm strip lengths and a second for 7 24 cm strip lengths um 80 6465 70 p4 Optional IEF electrode strips If desired small rectangular pads cut from the IEF electrode strips can be placed at the ends of the rehydrated Immobiline DryStrips just under the electrodes Although electrode pads are not required for making electrical contact between the IPG strip and the electrode they can improve the quality of results The pads absorb excess water as well as proteins with pls that are outside the pH range of the IPG strip A Important Before proceeding make sure the Ettan IPGphor unit is placed on a level surface Instructions Rehydrate the Immobiline DryStrip gels with the gel side down in the appropr
9. f ltigt in einem Abrufsystem gespeichert oder in irgendeiner Form oder mit irgendwelchen Mitteln bertragen werden Italiano um 80 6465 70 pvi A Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attentamente l intero contenuto del presente manuale Il punto esclamativo all interno di un triangolo equi latero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allegata al prodotto Si prega di inviare eventuali commenti al presente manuale a Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilit Amersham Biosciences 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 Biosciences Amersham Biosciences non potr essere ritenuta responsabile di incidenti 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 2002 Amersham Biosciences Corp Tutti i diritti riserv
10. g IEF Two areas of metal plating on the bottom extend up the sides of the strip holder These plated areas make contact with the power supply contact pads 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 strip holders have a coating to minimize protein adsorption Because some cleaning agents can remove this coating clean the strip holders only with the Ettan IPGphor Strip Holder Cleaner as directed The strip holders are very fragile and should be handled with care Electrodes The movable electrode clips can be placed anywhere along the strip holder where the elec trode 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 cups Sample cups can be placed almost anywhere along the length of the Ettan IPGphor strip holder that is not blocked by a protrusion For proper sealing of the cup to the gel the feet of the sample cup must all rest on the bottom of the channel Cover 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 mak
11. iate volume of rehydration solution using the Immobiline DryStrip Reswelling Tray If the tray is not available strips can be rehy drated in a standard Ettan IPGphor Strip Holder Rehydration in the Cup Loading Strip Holder is not recommended The channel is too wide for the recom mended rehydration volume Any unused rehydration solution can be frozen overnight and used to check sample cup placement later IPG strip length cm rehydration volume pl 7 125 11 200 13 250 18 340 24 450 O Allow the strips to rehydrate overnight Position the strip holder on the Ettan IPGphor platform The pointed end of the strip holder should contact the anodic electrode area and the blunt end should contact the cathodic electrode area of the Ettan IPGphor separation platform Fig 2 Positioning 7 cm and 11 cm IPG strips um 80 6465 70 e p6 Q Transfer the strips to the Ettan IPGphor Cup Loading Strip Holder The IPG strips should be placed faceup in the tray with the anodic pointed end of the IPG strip toward the pointed end of the strip holder The strip must be positioned so that the gel extends into both plated contact regions of the strip holder For the 7 cm and 11 cm IPG strips the cathodic end must be approximately 1 5 cm from the end of the channel and about 1 cm from the end of the cathodic rails see Fig 2 Center the strip down the length of the strip holder channel Protrusions a
12. line DryStrip Reswelling Tray 7 18 cm 1 80 6371 84 Immobiline DryStrip Reswelling Tray 7 24 cm 1 80 6465 32 Immobiline DryStrip Cover Fluid 1 liter 17 1335 01 IEF electrode strips 100 18 1004 40 Ettan IPGphor Strip Holder Cleaner 950 ml 80 6452 78 um 80 6465 70 e p14 Ettan Immobiline IPGphor and Drop Design are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Amersham Biosciences Corp 2002 All rights reserved All goods and services are sold subject to the terms and condi tions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Printed in the USA Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences AB SE 751 84 Uppsala Sweden Amersham Biosciences Corp 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA www amershambiosciences com Amersham Biosciences
13. long the sides guide the strip approximately straight although some manual adjustment of the strip may be necessary electrode contact Overlay Immobiline DryStrip Cover Fluid across the surface of the IPG strip It is important to distribute the oil evenly across the IPG strip and down the entire length of the holder Use only enough fluid to completely cover the strip without overfilling 3 5 ml Fig 3 Placing a filter paper pad at the end of the gel strip Fig 4 Seat the electrode on the pad at the end of the gel strip Optional Cut electrode pads about 5 mm long from IEF electrode strips Two pads per strip holder are required Wet the pads with deionized water and blot them almost completely dry Longer pads can be used if desired If using longer pads one end of the pad should overlap the end of the gel on the IPG strip The electrode must contact the other end of the pad 6 Place pads on both ends of the IPG strip Fig 3 Slide an electrode down on top of each pad Fig 4 Depending on the thickness of the IEF wick the electrode may not feel solid on top of the filter paper Pressure applied by the cover however will ensure complete contact when the lid of the Ettan IPGphor unit is closed Note For basic IPG strips superior focusing patterns are generally obtained when the sample cup is placed as close to the anodic electrod
14. r vus pour s assurer qu il est conforme aux sp cifications et normes en vigueur La garantie incluse dans les condi tions de livraison n est valable que si le produit a t install et utilis conform ment aux instructions fournies par Amersham Biosciences La soci t Amersham Biosciences 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 risques ayant un rapport avec l utilisation du produit mais sans aucune limitation quant a la nature de ces dommages 2002 Amersham Biosciences Corp 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 piii Espafiol A um 80 6465 70 piv Informacion importante para el usuario Para comprender el producto y utilizarlo con seguri dad es necesario leer este manual en su totalidad El signo de admiraci n en un tri ngulo equilatero en el manual advierte al usuario sobre la presencia de instrucciones importantes de oper
15. t has been installed and used according to the instructions supplied by Amersham Biosciences Amersham Biosciences shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related expo sures however caused arising from the faulty or incorrect use of the product O 2002 Amersham Biosciences Corp 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 Francais A Renseignements importants d utilization Pour une bonne compr hension et une utilisation en s curit maximale il convient de lire enti rement ce manuel Dans la documentation qui accompagne l instrument un point d exclamation dans un triangle quilat ral a pour but d attirer l attention de l utilisateur sur des instruc tions importantes de fonctionnment ou de maintenance Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser a Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Amersham Biosciences garantit l utilisateur que le produit livr a subi avec succ s tous les essais p
16. trips turn white and opaque after focusing Water evaporation If the strips are not covered properly with fluid the strips will lose moisture and urea will crystallize Make sure the recommended volume of cover fluid is spread across the entire length of the IPG strip Strips do not achieve 8000 V Higher conductivity of the rehydration solution Higher conductivity of the sample Short length IPG strips um 80 6465 70 e pl2 With higher concentration of IPG buffers or carrier ampholytes the IPG strips are more conductive than are strips with lower concentrations This may increase the time to reach 8000 V and complete focusing because the current is limited to 50 pA per strip in the Ettan IPGphor unit Using 0 5 IPG buffer is recommended for higher voltages High conductivity can also arise from the use of poor quality urea or other denaturants Urea is also prone to decompose to charged breakdown products Salts and ionic impurities in the sample can raise the conductivity of the strip 7 cm strips will not reach 8000 V The distance between the electrodes is shorter so that the volt age gradient V cm required to reach the 50 pA current limit is reached at a lower overall voltage A Important Do not use strong acids bases ketones alcohols or other reagents to clean the covers sample cups or electrodes or the parts may be damaged Cups may be briefly rinsed with ethanol if desired A
17. user manual 2 D electrophoresis Ettan IPGphor Cup Loading Strip Holder Ettan IPGphor isoelectric focusing system al Amersham um 80 6465 70 Rev BO 06 02 o e Biosciences Page finder Ettan IPGphor Cup Loading Strip Holder 1 INSETUCHONS siae 5 Suggested run conditions 11 Troubleshooting 12 Care and cleaning 13 Customer service information 13 Ordering information 14 English um 80 6465 70 pii A Important user information Please read this entire manual to fully understand the safe and effective use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of impor tant operating and maintenance instructions in the literature accompanying the instrument If you have any comments on this manual please send them to us at Amersham Biosciences SF Corp 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make changes in the specifications without prior notice Warranty and liability Amersham Biosciences 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 produc
18. ve rehydrated properly Examine the strip closely for a thin region in which the cup may not be able to seal properly Be sure to use the proper volume of rehydration solution for a sufficient time to allow complete rehydration Briefly centrifuge the protein sample prior to loading to remove insoluble material Particulates and insolu ble material will impede sample entry and result in vertical streaks in the second dimension gel Pipette up to 100 ul of sample into the sample cup Fig 7 Place the cover over the assembled strip holder um 80 6465 70 e p10 Place the cover over the strip holder Fig 7 Close the Ettan IPGphor cover O Program the Ettan IPGphor with the desired run para meters Ramping the voltage slowly while the sample is entering the IPG strip will improve results Optimal ramp voltages and times or Vh volt hours totals must be determined empirically for each sample type Focusing after sample cup application frequently requires fewer Vh than in gel sample rehydration load ing methods particularly on basic pH range strips Suggested run conditions 50 pA per strip 20 C voltage step duration step duration voltage gradient type h min Vh 1 500 Gradient 0 01 2 4000 Gradient 1 30 3 8000 Step n hold as per below 7 cm 3 10 L 3 10 NL 4 7 L 6 11 0 5 1 h t 3700 VI lcm 3 10 L 4 7 L 1 3 3 h t 10600 V lcm
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