TE 22 Mighty Small™ Transphor Tank Transfer Unit User Manual
Contents
1. Bjerrum O J Larsen K and Heegaard N CRC Handbook of Immunoblotting of Proteins Vol 1 Section 7 CRC Press 1988 Burnette W N Western blotting electrophoretic transfer of proteins from sodium dodecyl sulfate poly acrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiod inated protein A Anal Biochem 112 195 1981 Gallagher S Winston S E Fuller S A and Hurrell J G R Immunoblotting and Immunodetection In Current Protocols in Molecular Biology 10 8 1 10 8 17 Greene Publishing and Wiley Interscience NY 1993 Gershoni J M Davis F E and Palade G E Protein blotting in uniform or gradient electric fields Anal Biochem 144 32 40 1985 Gershoni J M and Palade G E Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacry lamide gels to a positively charged membrane filter Anal Biochem 124 396 405 1982 Gershoni J M and G E Palade 1983 Protein Blotting Principles and Applications Anal Biochem 131 1 15 Gibson W Protease facilitated transfer of high molecular weight proteins during electrotransfer to nitro cellulose Anal Biochem 118 1 1981 Lin W and Kasamatsu H On the electrotransfer of polypeptides from gels to nitrocellulose membranes Anal Biochem 128 302 311 1983 Matsudaira P Sequence from Picomole Quantities of Proteins Electroblotted onto Polyvinylidene Difluoride Membranes J Biol Chem 262. other hydrophobic membranes in methanol Then soak all membrane types in transfer buffer for 2 5 minutes Open the cassette by releasing both latch tabs along the edge opposite the hinges Place the opened cassette into a tray filled with at least 3 cm of transfer buffer Assemble the transfer stack so that molecules will migrate toward the membrane For negatively charged macromolecules such as nucleic acids and most proteins build the stack on the grey half of the cassette and then later position the lid so that the grey side faces the red lead or anode Place one 3 mm thick foam sponge on the opened submersed cassette and press gently until all air is expelled Place one sheet of blotting paper on the sponge and then place the membrane on the blotting paper Place the gel which con tains a sample that has been electrophoretically separated and equilibrated if required with transfer buffer on the membrane Gently roll a glass pipet or test tube over the gel to expel trapped air between the membrane and gel Cover the gel with a sheet of blotting paper and then place a sponge of the proper thick ness see the diagram below again pressing gently to expel trapped air Close the cassette and press lightly to lock the tabs The assembled cassette should hold the gel in firm contact with the membrane without squeezing the gel If the stack seems loose add sheets of blotting paper if the stack seems tight replace the top spon
3. tions 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 pr vus pour s assurer qu il est conforme aux sp cifications et normes en vigueur La garantie incluse dans les con ditions 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 utilisa tion incorrecte ou non approuv e du produit ou d coulant de cette utili sation 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 la nature de ces dommages Copyright 1997 Amersham Biosciences Tous droits r serv s La reproduction le stockage dans un syst me de r cup ra tion 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 Espa ol Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguridad es necesario leer este manual en su totalidad El signo
4. Tris cannot be used as in pep tide sequencing CAPS can improve transfer because of its effect on the charge of the protein see Matsudaira 1987 For native proteins we suggest using the electrophoresis buffer for transfer as well Use the Towbin buffer to transfer SDS denatured proteins toward the anode Towbin buffer 25 mM Tris 192 mM glycine 20 v v methanol pH 8 3 2 liters Tris FW 121 1 25 mM 6 0g Glycine FW 75 07 192 mM 28 8 g SDS FW 288 4 0 1 3 5 mM 2 0g Dissolve in 1 5 liters distilled water Add methanol as required Bring to 2 liters with distilled water Do not adjust the pH which should be between 8 2 and 8 4 Optional Chill before use Optional Adding SDS can improve transfer efficiency PDepending on the membrane type selected adding methanol can improve the transfer results see dis cussion and table above Because buffers containing methanol may deteriorate if stored for long periods add methanol as required just prior to transfer CAPS buffer 1X 10 mM CAPS pH 11 0 2 liters CAPS FW 221 3 10 mM 444g 3 cyclohexylamino 1 propanesulfonic acid Dissolve in 1 5 liters distilled water adjust to pH 11 0 with conc HCI Adjust volume to 2 0 liters TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Current Protocols in Molecular Biology 1993 A 2 1 PSambrook J et al 1989 Molecular Cloning A Laboratory Manual B 23 Nucleic acid transfers Nucleic acids
5. and the black lead into the black output jack In most systems the red lead is the anode and the black lead is the cathode Cooling is strongly recommended Any setting that results in higher than 5 W of power will generate enough heat to require active heat control A refrigerated circula tor bath using water should be set to about 10 C If using 50 50 ethylene glycol water the temperature can be set lower Chill the buffer before use if possible Set the power supply Constant current mode is recommended If constant voltage mode is selected carefully monitor the current increased current increases Joule heating If the current exceeds 0 4 A decrease the voltage Protein Nucleic acids Buffer Towbin 1X TBE Current A 0 4 0 3 Voltage V 100 50 Transfer time 1 hour 1 hour Coolant temp 10 C 10 C or less If available set the power supply timer Most transfers are complete within one hour but larger molecules or thicker gels may require longer transfer times the optimum transfer time for each system must be determined empirically TE 22 Mighty Small Transphor Tank Transfer Unit User Manual After the transfer is complete 1 Note It is a good idea to stain the gel to determine the com pleteness of the transfer Note If the buffer will be reused store it in a separate contain er and cool to 10 C before reuse Turn the voltage and current settings to zero and turn off the power supply Disco
6. by Amersham Biosciences TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Tank transfer unit main components AN ___ Color coded leads A power supply capable of delivering up to 100 V and 400 to 500 mA is required _ Transfer tank TT Up to four cassettes fit into the slots Electrode retaining screw 2 _ __ Fill levels Cassette hook _ Heat exchanger ports 2 Included but not shown g Gel cassettes 4 Foam sponges 6 mm thick 4 Foam sponges 3 mm thick 8 Blotting paper sheets 25 Heat exchanger pressure safety valve TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Important information The safety lid must be in place before connecting the power leads to a power supply Turn all power supply controls off and disconnect the power leads before removing the safety lid with distilled water before use A Informations importantes Le couvercle de s curit doit tre en place avant de brancher les prises au g n rateur Eteindre le g n rateur et d brancher les prises avant d enlever le couvercle de s curit Rinser seulement les lectrodes pas les banana plugs avec de l eau distill e juste avant l utilisation Rinse only the electrodes not the banana plugs Circulate only water or 50 50 water ethylene gly col through the heat exchanger Never introduce anti freeze or any organ
7. remove buffer as required so that the level falls between the minimum and maximum buffer level lines Buffer above the maxi mum buffer level line may cause corrosion of the electrical contacts Final assembly and transfer 1 Install the lid The cassettes are color coded to match the leads in the lid To transfer toward the anode orient the lid so that the grey half of the cassette faces the anode or red lead and the black half of the cassette faces the cathode or black lead Make sure the banana plugs seat into the connectors in the lid Note Take care in orienting the lid so that all species migrate toward the membrane when the electric field is applied The migration direction depends on both the characteristics of the sample and the pH of the transfer buffer If the species of interest is negatively charged in the transfer buffer and the stack is assembled so that the membrane is nearest the grey side of the cassette then this side faces the anode Most proteins migrate toward the anode in the Towbin Tris glycine methanol buffer system indepen dent of the presence of SDS and under most conditions nucleic acids are negatively charged and also migrate toward the anode Use only an approved power supply such as the Hoefer EPS 2A200 Make sure the power supply is off and all controls are set to zero Plug the color coded leads from the lid of the transfer unit into the power supply the red lead into the red output jack
8. 2 10035 1987 Ohmsted J B Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples J Biol Chem 256 11955 1981 Renart Reiser J and Stark G R Transfer of proteins from gels to DBM paper and detection with antisera a method for studying antibody specificity and structure Proc Natl Acad Sci USA 76 3116 1979 Sambrook J et al Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press B 23 1989 Southern E M Detection of specific sequences among DNA fragments separated by gel electrophoresis Molec Biol 98 3 503 517 1975 Stellway E J and Dahlberg A E Electrophoretic transfer of DNA RNA and protein onto DBM paper Nucleic Acids Res 8 299 1980 Symington J Green M and Brackmann K Immunological detection of proteins after electrophoretic transfer from gels to diazo paper analysis of adenovirus encoded proteins Proc Natl Acad Sci USA 78 177 181 1981 Towbin H Staehelin T and Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications Proc Natl Acad Sci USA 76 4350 4354 1979 TE 22 Mighty Small Transphor Electrophoresis Unit Instructions Customer Service Information Technical Service and Repair Amersham Biosciences offers complete technical support for all our prod ucts If you have any questions about how to use this product o
9. A M E R S H A M B los G It EN CG E S TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Transfer Unit Function and Description 1 Specifications 2 Important Information 4 Operating Instructions 5 Care and Maintenance 8 Troubleshooting 9 Electrotransfer Notes 11 Bibliography and References 15 Customer Service Information 16 Amersham 80 6245 68 TE22 IM Rev C1 4 97 Biosciences TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Renseignements importants d utilization Pour une bonne compr hension et une utilisa tion en s curit maximale il convient de lire enti rement ce manuel Francais 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 instructions impor tantes de fonctionnement ou de maintenance Le symbole de l clair dans un triangle quilat ral a pour objet d attirer l attention de l utilisateur sur un danger d ex position la haute tension Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser A mersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d effectuer des modifica
10. a de recuperaci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa Wichtige Benutzerinformationen F r ein vollst ndiges Verst ndnis und eine De utsch sichere Handhabung dieses Produktes ist es notwendig daf 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 Ein Blitzsymbol in einem gleichseitigen Dreieck soll den Benutzer auf die Gefahr anliegender Hochspannungen hin weisen Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Amersham Biosciences Inc Marketing Department 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 ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uterten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem den von Amersham Biosciences gelieferten Anweisungen instal liert und benutzt wurde Amersham Biosciences bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbu en Einko
11. ces en plas tique sont pr vues pour r sister une temp rature constante de 45 C Faire circuler l eau dans l changeur vertical pour minimiser l chauffement afin d viter des dom mages irr parables l instrument Ne pas connecter l changeur vertical circulation d eau un robinet ou quelque source de refroidissement dont la pres sion n est pas r guli re Pour des coulages plus long on peut aussi contr ler la temp rature en refroidissant le tampon avant I util isation et ou en utilisant l instrument dans une chambre froide Utiliser uniquement la quantit prescrite d ponges et de papier filtre afin que la cassette ne soit pas trop pleine Trop de materiels peut endommager la cassette Si l instrument n est pas utilis en conformit avec les recommandations du fabriquant les protections de s curit qui quipent cet appareil peuvent tre ren dues in fficaces Seulement les accessoires et pi ces detach es approuv s ou fournis par Amersham Biosciences sont recommand s pour l utilisation l entre tien et r paration de cet appareil TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Note For quick and easy connec tions install Quick disconnect fittings with valves in the line Operating Instructions Transfer the sample as soon as possible after electrophoresis to avoid sample dif fusion into the gel Each step is described below Prepare the buffer Prepare a
12. de admiraci n en un tri ngulo equil tero en el manu al advierte al usuario sobre la presencia de instrucciones importantes de operaci n y mantenimiento del aparato 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 amabili dad de remitirlo a Amersham Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el derecho a modificar las especi ficaciones 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 especifica ciones 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 consecuencias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del producto CopyrightO 1997 Amersham Biosciences Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistem
13. e can be controlled by circulating cooled liquid through the heat exchanger in the base The buffer is separated from the coolant by a heat conducting alumina plate Unpacking Unwrap all packages carefully and compare contents with the packing list mak ing sure all items arrived If any part is missing contact Amersham Biosciences Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediate ly Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Specifications Gel size Max wattage Max voltage Max amperage Max temperature Buffer required Environmental operating conditions Installation category Pollution degree Dimensions w x h x d Product certifications TE 22 Mighty Small Transphor Tank Transfer Unit User Manual up to four 9x10 cm gels 50 W 100 V 500 mA 45 C 1 5 liters depending on the number of cassettes in place Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Il 2 14x24x16 5 cm 5 5x9 5x6 5 in EN61010 1 UL3101 1 CSA C22 2 1010 1 CE This declaration of conformity is only valid for the instrument when it is p used in laboratory locations p used as delivered from Amersham Biosciences except for alterations described in the User Manual and p connected to other CE labeled instruments or products recommended or approved
14. ersham 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 specifica tions 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 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 exposures however caused arising from the faulty and incorrect use of the product Copyright 1997 Amersham Biosciences 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 TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Transfer Electrophoresis Unit Function and Description The Hoefer TE 22 Mighty Small Transphor Electrophoresis unit rapidly transfers proteins DNA or RNA from up to four small format polyacrylamide or agarose gels onto a membrane Gels and membranes are held by a cassette which is sub merged into the transfer tank Molecules migrate under an electric field to the membrane where they are bound The transfer buffer temperatur
15. ge over the gel with a sheet of blotting paper If you remove the bottom sponge below the gel substitute at least two sheets of blotting paper to create space between the membrane and the cassette panel one 3 mm sponge for gels gt 1 5 mm _ one 6 mm sponge ae for gels lt 1 5 mm LE ___Blotting paper ly Cel Membrane a Blotting paper one 3 mm sponge Assemble the cassette in a tray contain ing transfer buffer about 3 cm deep TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Important note Never allow the buffer temperature to exceed 45 C Excessive heat will cause the unit to warp Typical transfer parameters Parameters for your sample and buffer system must be determined empirically Install the cassette s 1 For only one or two gels choose the cassette positions nearest the center The cas settes must be oriented so that the hinge side is facing up and all black panels of the cassettes are facing the same side of the transfer unit Work quickly when moving the assembled cassette s to the tank to avoid draining the sponges Place the tray holding the cassette s near the tank lift out one cas sette at a time and slide it into a set of vertical slots Do not discard the buffer Once in place tap the cassette lightly until most air bubbles are dislodged A few small bubbles in the sponges are unlikely to interfere with the transfer Inspect the buffer level Add or
16. ic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperature above 45 C All plastic parts are rated for 45 C contin uous duty Circulate coolant through the heat exchanger to minimize heating Overheating will cause irreparable damage to the unit Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated For longer runs you can control heating some what by chilling the buffer before use running the unit in a cold room or both When assembling the transfer cassette use only the required amount of gel support materials sponges and blotting paper to prevent over stuffing the cassette Excess materials may result in cassette damage If this equipment is used in a manner not speci fied by the manufacturer the protection provid ed by the equipment may be impaired Only accessories and parts approved or supplied by Amersham Biosciences may be used for operating maintaining and servicing this product Faire circuler seulement de l eau ou 50 50 d eau et d thylene glycol dans l changeur vertical cirula tion d eau Ne jamais utiliser d anti gel ou tout autre solvant organique avec cet instrument Les solvants organiques causeraient des dommages irr parables l appareil Ne pas utiliser avec un tampon une temp ra ture au dessus de 45 C Toutes les pi
17. izioni 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 Copyright 1997 Amersham Biosciences 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 TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Important user information E li h Please read this entire manual to fully under n g IS stand 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 important oper ating and maintenance instructions in the literature accom panying the instrument The lightning symbol within 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 Biosciences Inc Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Am
18. lectrophoretic transfer can improve transfer efficiency over non electrophoretic blotting especially for proteins but no quantitative transfer technique has yet been developed due to the complexity of the reactions Quantitative recovery is actually not required for most purposes because binding macromolecules to a membrane increases the sensitivity of detection methods such as autoradiogra phy and permits detection of specific proteins by antibodies or affinity labels and of specific nucleic acids by hybridization with complementary strands of RNA or DNA The buffer can be chosen to result in a transfer toward either the cathode or the anode The buffer pH must be such that all species of interest are charged and migrate in the same direction The ionic strength should not be too high since this will produce excessive current and heat For this reason the high salt condi tions used by Southern for capillary blotting of DNA cannot be used The most widely used buffer systems are those of Towbin et al for transferring proteins and of Bittner et al for transferring nucleic acids Buffer systems for transfer of each type of sample are listed later in this section Factors affecting the transfer Parameters such as sample characteristics membrane type gel pore size and the transfer buffer used all contribute to the transferability of macromolecules and should be kept in mind when developing a protocol Very small molecular species for insta
19. lete transfer Blank areas on the membrane Y Y Remove all trapped air pockets in the transfer stack assembly assemble the stack while it is submerged in transfer buffer gently press on each sponge as it is added to the stack and roll a glass pipette or test tube over the membrane and gel to eliminate all air bubbles Reduce the stirring speed to prevent turbulence Process only one strip or membrane in each tray or cassette to prevent over lapping Use buffer with a lower ionic strength Check electrode continuity During the transfer a continuous stream of gas is released along the entire length of the electrodes If bubbles do not form along the entire length of the electrode replace the electrode If cassettes are bowed when empty replace Overpacking the cassette caus es it to bow see the recommended assembly instructions on page 6 Grid pattern on membrane C4 Add extra sheets of blotting paper to increase the clearance between the cassette panel and the gel Take care not to overstuff the cassette the gel should be held firmly and evenly between the sponges but not so tightly that it is squeezed Molecules do not migrate out of gel SK LN Vs Increase the field strength Increase transfer period Try doubling it Do not use staining or fixing agents on the gel before transfer Use a thinner gel Reduce the gel acrylamide concentration Check that the buffer pH is close to the intended pH Most buffe
20. minimum of 1 5 liters of the appropriate transfer buffer Refer to the Electrotransfer Notes section for a discussion of membranes and buffers Chill the buffer before use if possible Prepare the unit 1 Rinse the the unit and cassettes with distilled water 2 Active cooling is optional but strongly recommended If no active cooling will 3 4 be used go to step 3 Note Connect the heat exchanger to a circulator bath such as the MultiTemp Ill Circulate only water or 50 50 water ethylene glycol to prevent damage to the unit The circulator pump must not generate a pressure greater than 0 7 bar 10 psi above atmospheric pressure Set the temperature to 10 C or higher if circulating only water If using 50 50 ethyl ene glycol water the temperature can be set lower Start the circulator bath at the same time as the transfer First attach tubing to the red pressure relief valve between the water inlet and outlet ports and insert the free end into the bath or other container or drain to catch any pressure relief overflow The relief valve opens if the pressure within the heat exchanger exceeds 10 psi Prepare two lengths of 9 mm 38 vinyl or silicone tubing Slide hose clamps 4 total onto each end of two lengths of tubing Attach one end of each length of tubing to a heat exchanger port Attach the free ends of each length of tubing to the circulator bath ports one to the inlet and the other to the outlet Secure
21. mmensverluste 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 Copyright 1997 Amersham Biosciences 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 Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attentamente l intero contenuto del presente manuale Italiano Il punto esclamativo all interno di un triangolo equilatero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allega ta al prodotto 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 Biosciences Inc Marketing Department 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 cond
22. nce migrate quickly but often do not bind as well as larger mol ecules large molecules bind more efficiently but do not elute from the gel as rapidly The rate of elution is also affected by the pore size of the gel and the ori entation of the molecules Further the degree to which molecules bind to the membrane is influenced by membrane characteristics such as pore size and type and buffer characteristics such as pH salt type and concentration and the presence of detergents such as sodium dodecyl sulfate SDS Conditions required for efficient elution may not coincide with optimal conditions for binding To find the optimum conditions for transferring your sample balance these effects If the sample elution rate is TE 22 Mighty Small Transphor Tank Transfer Unit User Manual slow a longer transfer period may be required In our experience low voltage transfers for longer periods do not offer much improvement If sample binding is inadequate try different buffer conditions For a comprehensive review see Gershoni and Palade 1983 If the transfer buffer system is different from the electrophoresis buffer system the gel should be equilibrated with the transfer buffer before the transfer to ensure swelling or shrinking occurs before the gel contacts the transfer mem brane If this step is skipped band distortion or loss of resolution could result Instrument guidelines Cooling Considerable Joule heat is generated during any
23. ne Chemical parameters Y Y Y Fix or crosslink the molecule onto the membrane according to the require ments of the nucleic acid protein or membrane type Prepare protein transfer buffer without SDS Verify the optimal amount of methanol required for the membrane type and check the buffer solution Add 10 20 methanol to the transfer buffer to enhance binding to nitrocellulose Membrane parameters Y Y Y Y K Wear gloves when handling membranes Store membranes at ambient temperature out of direct sunlight to keep the membranes activated Use a membrane with a smaller pore size 0 10 0 20 um if proteins pass through the membrane or use a different membrane type Place a membrane both over and under the gel if you suspect one protein is moving in the opposite direction from the majority of the proteins Check both membranes for protein s Check if too much sample is available for the binding surface area by apply ing two membranes instead of one If blow through occurs reduce the sample load TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Electrotransfer Notes Electrophoretic transfer advantages Electrophoretic transfer of proteins and nucleic acids is much faster than the blotting methods first described by Southern for DNA Alwine et al for RNA or Renart et al for proteins The tank transfer method uses high current to reduce the transfer time of most samples to 45 60 minutes E
24. ng to nitrocel lulose Methanol improves binding in part by removing protein bound SDS In the absence of methanol labeled bovine serum albumin BSA passes through at least five layers of membranes Methanol may cause a gel to shrink however so TE 22 Mighty Small Transphor Tank Transfer Unit User Manual the elution rate decreases By using a cationic membrane such as nylon which binds the proteins more efficiently and omitting methanol from the transfer buffer Gershoni and Palade obtained a much more quantitative transfer The disadvantage of cationic membrane is that protein stains also bind well so that the staining background tends to be very high Properly quenched however this paper can be used for antibody detection or other overlay methods of pro tein identification A summary of membrane type and recommended methanol concentration follows Membrane type Methanol Charged nylon 0 Nitrocellulose lt 20 PVDF lt 15 Some workers have reported to us that a low concentration of SDS 0 1 improves the transfer of protein from an SDS gel Burnette 1981 and Symington et al 1981 investigated the effect of the molecular weight of pro tein Gibson 1981 describes a method to increase the extent of transfer of large proteins by limited cleavage with pronase during transfer Protein transfer buffers Use a buffer with low ionic strength such as the two listed below to prevent overheating Use the alternate CAPS buffer when
25. nnect the leads from the power supply jacks Lift off the lid Use the plastic hook stored in the holder at the side of the unit to lift up a cassette just far enough to be able to grab it Open each cassette carefully and remove the gels and membranes Label each membrane and indicate the sample side Lift membrane s with blunt forceps and air dry or follow the instructions accompanying your protocol Discard the blotting paper but reuse the sponges Rinse the unit immediately after use See the Care and Maintenance Section below Care and Maintenance Cleaning Do not autoclave or heat any part above 45 C Do not expose to organic solvents Never use abrasive detergents If using radioactive reagents decontaminate the unit with a cleaning agent such as Count off Rinse the tank cassettes and sponges with distilled water immediately after each use Allow the unit to air dry completely Periodically wash with a dilute solution of a mild detergent Removing the electrode panel s For more thorough cleaning or to replace damaged electrodes remove each elec trode panel by unscrewing the retaining screw far enough to allow the panel to slide out Use the hook on the side panel to pull the electrode panel up do not pull the panel up by the banana plug Take care to not stretch or break the plat inum wire when handling the panel TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Troubleshooting Incomp
26. normally must be transferred in denatured form for most efficient binding RNA is normally denatured with glyoxal before separation or separated in denaturing gels containing formaldehyde or methyl mercury However dou ble stranded DNA is usually denatured in the gel with NaOH The alkali must be neutralized and the gel equilibrated in transfer buffer before electrotransfer For both DNA and RNA gels any SDS must also be removed to assure efficient bind ing Bittner et al 1980 wash gels three times 20 minutes each to assure com plete removal of denaturants and detergents See Bittner et al for a study of the transfer efficiency for DNA of different sizes The Bittner transfer buffer contains 25 mM sodium phosphate pH 6 5 Also described is a method for the introduction of nicks by limited nuclease action in order to facilitate transfer of larger DNA fragments Recommended DNA buffers include the Bittner sodium phosphate buffer see reference and TBE For RNA TAE is recommended TBE and TAE stock recipes are listed below These buffers are most often diluted to 1X but the concentra tion can range down to 0 1X Cooling is strongly recommended for these buffers especially at higher concentrations EDTA solution 0 5 M EDTA pH 8 0 100 ml Na EDTA 2H 0 FW 372 2 0 5 M 18 6g Dissolve in 70 ml distilled water Adjust to pH 8 0 with 10 M NaOH approx 5 ml then add distilled water to 100 ml DNA transfer buffer 10X 10X Tri
27. r would like to arrange to repair it please call or fax your local Amersham Biosciences representative Important Request a copy of the Amersham Biosciences Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Printed in the USA Ordering Information TE 22 Mighty Small Transphor Electrophoresis Unit Includes 4 gel cassettes 8 foam ponges 3 mm thick 4 foam sponges 6 mm thick 25 sheets of blotter paper Accessories and Replacement Parts Qty 1 Gel cassette 2 foam sponges 3 mm thick and 1 foam sponge 6 mm thick 1 Foam sponges 9 x 10 5 cm 6 mm thick Foam sponges 9 x 10 5 cm 3 mm thick Electrode Panel Safety lid with cables High voltage leads with jacks Quick fit coupler body female to fit 9 5 mm 3 8 ID tubing Quick fit coupler body male to fit 9 5 mm 3 8 ID tubing Tubing for coolant silicone 8 12 mm Blotter Paper Blotter paper sheets 7 x 8 cm Blotter paper sheets 9 x 10 5 cm Transfer Membranes Nitrocellulose 0 45 um pore size Nitrocellulose sheets 9 x 10 5 cm Nitrocellulose roll 33 cm x 3 m 0 2 um pore size Nitrocellulose roll 33 cm x 3 m Nylon 0 45 um pore size Nylon 66 Standard roll 33 cm x 3 m Nylon Standard GeneBina roll 20 cm x 3 m Nylon 66 Plus chargea roll 33 cm x 3 m Companion Prod
28. rs should not be titrated make fresh buffer Use 3 5 mM SDS 0 1 in the transfer buffer Avoid including methanol in the transfer buffer or reduce the amount to the absolute minimum Use reagent grade chemicals Increase the length of time Southern blots are depurinated Increase the net charge on the protein by changing to a transfer buffer with a different pH Lower pH lt 6 7 increases the positive charge on proteins higher pH gt 6 7 increases the negative charge on proteins Continued TE 22 Mighty Small Transphor Tank Transfer Unit User Manual For more troubleshooting hints refer to Bjerrum O J et al 1988 Diffuse band patterns Y Y Transfer immediately after electrophoretic separation If equilibrating before the transfer shorten or eliminate the equilibration time or move the gel to the cold room during equilibration If transfer buffer contains methanol gt 10 equilibrate the gel in transfer buffer for 30 minutes to allow it to shrink before assembling the stack Note Because methanol causes the gel to shrink slightly large molecules may migrate more slowly Take care that the gel is held firmly against the membrane and that it does not shift once contact is made If excess heating occurs during the transfer lower the temperature of the cooling fluid in the heat exchanger Check that the preferred binding surface of the membrane if any contacts the gel Inefficient binding to membra
29. s borate EDTA TBE pH 8 2 1 liter Tris FW 121 1 900 mM 109 0 g Boric acid FW 61 83 900 mM 55 6 g EDTA solution 0 5 M pH 8 0 20 mM 40 0 ml Distilled water to 1 0 liter Do not adjust pH Dilute to 1X before use to yield 90 mM Tris 90 mM boric acid and 2 mM EDTA This dilution is com monly used but dilutions down to 0 1X may be used should it be necessary to decrease the amount of current in the system in order to control overheating RNA transfer buffer 10X 10X Tris acetate EDTA TAE pH 8 4 1 liter Tris FW 121 1 400 mM 48 4 g Acetic acid glacial 17 4 M 200 mM 11 4 ml EDTA solution 0 5 M pH 8 0 10 mM 20 0 ml Distilled water to 1 0 liter Do not adjust pH Dilute to 1X before use to yield 40 mM Tris 20 mM acetate and 1 mM EDTA This dilution is com monly used but dilutions down to 0 1X may be used should it be necessary to decrease the amount of current in the system in order to control overheating TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Bibliography and References Alwine J C Kemp D J and G R Stark Method for detection of specific RNAs in agarose gels by transfer to DBM paper and hybridization with DNA probes Proc Natl Acad Sci USA 74 5350 5354 1977 Bittner M Kupferer P and Morris C F Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets Anal Biochem 102 459 471 1980
30. the connections with the hose clamps Place do not drop a magnetic stirring bar in the buffer tank Dropping objects into the tank may crack the alumina plate Set the unit onto a magnetic stirrer and fill transfer buffer to the Start fill level line on the front of the tank This requires approximately 0 7 liters Set the stirrer to low medium which accomplishes buffer circulation without forcing buffer through the cassettes Note Even if no cooling is required for your system the buffer should be circulated with a stirrer to avoid buffer depletion at the electrodes TE 22 Mighty Small Transphor Tank Transfer Unit User Manual Note Always wear gloves when handling membranes to avoid getting fingerprints on them Important Take great care in removing all air bubbles at each step because the presence of air bubbles especially between the membrane and gel blocks transfer Transfer stack assembly The stack is oriented so that negatively charged molecules migrate toward the grey anode Note Try to place the gel correctly the first time because pro teins may begin to transfer immediately once transfer has begun moving the gel will distort results or cause shadow bands on the blot Assemble the transfer cassette 1 Pre wet nitrocellulose or nylon membranes with distilled water Pre wet PVDF or The cassette panels are color coded black top cathode side grey bottom anode side OR
31. transfer because of the high cur rent employed so active cooling is recommended especially for transfers requir ing more than one hour protein transfers where biological activity must be retained or transfer of nucleic acids The high conductivity of the phosphate buffer used by Bittner et al 1980 leads to a relatively rapid temperature rise Buffer temperature should not exceed 45 C because the cassettes and electrode supports may warp Use a circulator bath set to 10 C if using water as a coolant You can use a lower setting if the coolant is 50 50 ethylene glycol water Never leave the unit unattended for more than one hour under high power conditions gt 250 mA Power setting If using a power supply that can be set to either constant current or constant voltage mode we recommend that it be set to operate in constant current mode Buffer conductivity increases with temperature During blotting in an uncooled chamber Joule heating and rising conductivity may result in danger ous overheating if the power supply is set to maintain constant voltage If a constant voltage power supply must be used monitor and adjust the voltage to maintain a current at or below 400 mA Protein transfers Study summaries Gershoni and Palade 1982 investigated factors affecting protein recovery from SDS gels to nitrocellulose or DBM paper According to their findings methanol in the Towbin buffer system is necessary to achieve efficient bindi
32. ucts Hoefer EPS 24200 Power Supply 115 V 230 V Hoefer HB 1100D Red Roller II 115 V 230 V Hoefer HB 400 Mini Hydribization Oven 115 V 230 V 25 50 10 Code No 80 6204 26 80 6204 64 80 6205 02 80 6205 21 80 6204 45 80 6205 78 80 6177 09 80 6115 15 80 6115 53 80 1106 56 80 6211 48 80 6205 40 80 6221 17 80 6221 55 80 6220 22 80 6221 93 80 1247 87 80 6221 74 80 6274 18 80 6274 37 80 6038 96 80 6244 92 80 6041 81 80 6242 00
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