Hoefer™ TE 22
Contents
1. Electrophoretic 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 Quantita tive recovery is actually not required for most purposes because binding macromolecules to a membrane increases the sensitivity of detection methods such as autoradiography 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 conditions 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 epl5 e pl6 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 s2. CA 94107 USA support hoeferinc com Hoefer Inc beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern Gew hrleistung and haftung Hoefer Inc garantiert daB das gelieferte Produkt sorgf ltig auf die Einhaltung der ver ffentlichten Spezifikationen getestet wurde Die in den Lieferbedingungen n her erl uter ten Gew hrleistungsanspr che gelten nur dann wenn das Produkt gem den von Hoefer Inc gelieferten Anweisungen installiert und benutzt wurde Hoefer Inc bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewin neinbu en Einkommensverluste entgangene Gesch ftsab schl sse Verlust der Gebrauchsf higkeit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden Espa ol A A Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguridad es necesario leer este manual en su totalidad El signo de admiraci n en un tri ngulo equil tero en el manual advierte al usuario sobre la presencia de instruccio nes 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 amabilidad de remitirlo a Hoefer Inc 953 Indiana Street San
3. Francisco CA 94107 USA support hoeferinc com Hoefer Inc se reserva el derecho a modificar las especifica ciones sin previo aviso Garant a y responsabilidad Hoefer Inc garantiza que el producto entregado ha sido probado a fondo para comprobar el cumplimiento de las espe cificaciones publicadas La garant a incluida en las condicio nes de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Hoefer Inc Hoefer Inc 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 Italiano O pvi A 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 equilatero indica all operatore la presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione allegata 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 Hoefer Inc 9
4. However double stranded DNA is usually denatured in the gel with NaOH The alkali must be neutral ized and the gel equilibrated in transfer buffer before electrotransfer For both DNA and RNA gels any SDS must also be removed to assure efficient binding Bittner et al 1980 wash gels three times 20 minutes each to assure complete removal of denaturants and detergents See Bittner et al for a study of the transfer effi ciency for DNA of different sizes The Bittner transfer buffer contains 25 mM sodium phos phate 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 concentration can range down to 0 1X Cooling is strongly recommended for these buffers espe cially at higher concentrations EDTA solution 0 5 M EDTA pH 8 0 100 ml Na2EDTA 2H20 FW 372 2 0 5 18 6 g 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 Tris 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 adjus
5. do not pull the panel up by the banana plug Take care to not stretch or break the platinum wire when handling the panel Troubleshooting problem solution Incomplete transfer Blank areas on the membrane Grid pattern on membrane Molecules do not migrate out of gel 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 overlapping Use buffer with a lower onic 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 causes it to bow see the recommended assembly instructions on page 7 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 Increase the field strength Increase transfer period Try doubling it Do not use staining or fixing agents on the
6. 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 Fig 2 Transfer stack assembly The stack is oriented so that negatively charged molecules migrate toward the grey anode Important Do not overstuff the cassette Note Try to place the gel correctly the first time because proteins may begin to transfer immediately once transfer has begun moving the gel will distort results or cause shadow bands on the blot The cassette panels are color coded black top cathode side grey bottom anode side UTN NUM 4 O AL E AI O DO PES 4 A e p8 O YAA A sponge and then place the membrane on the blot ting paper Place the gel which contains 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 thickness see the diagram below again pressing gently to expel trapped air Q 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 o
7. that the preferred binding surface of the membrane if any contacts the gel Fix or crosslink the molecule onto the membrane according to the requirements 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 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 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 applying two membranes instead of one If blow through occurs reduce the sample load For more troubleshooting hints refer to Bjerrum O J et al 1988 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
8. 3 mm thick and 1 TE24 1 foam sponge 6 mm thick Foam sponges 9 x 10 5 cm 6 mm thick 4 TE25 Foam sponges 9 x 10 5 cm 3 mm thick 4 TE25F 1 8 Electrode panel 1 TE23 Safety lid with cables 1 TE29 High voltage leads with jacks pair SE6056 HV Quick fit coupler body female to fit 9 5 mm 3 8 ID tubing 2 QF3 8 Quick fit coupler body male to fit 9 5 mm 3 8 ID tubing 2 QFX3 8 Blotter paper Blotter paper sheets 9 x 10 5 cm 50 TE26 Companion products Hoefer PS 2A200 Power Supply 200 V 2A 1 PS2A200 e p24 Contrad 70 and Decon 90 are trademarks of Decon Lab Printed in the USA Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA www hoeferinc com Hoefer
9. 53 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Garanzia e responsabilit Hoefer Inc 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 Hoefer Inc Hoefer Inc non potr essere ritenuta responsabile di inci denti 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 Transfer Electrophoresis Unit function and description The Hoefer TE22 Tank Transfer 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 submerged into the trans fer tank Molecules migrate under an electric field to the membrane where they are bound The transfer buffer temperature 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 making sure all items ar
10. alcohols or organic solvents Alcohols or organic solvents will cause irreparable damage to the unit If this equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Use of lt 20 methanol methyl alcohol in transfer buffers is the only exception Francais Informations importantes e Le couvercle de s curit doit tre en place avant de brancher les prises au g n rateur e Eteindre le g n rateur et d brancher les prises avant d enlever le couvercle de s curit e Rinser seulement les lectrodes pas les banana plugs avec de l eau distill e juste avant l utilisation e Faire circuler seulement de l eau ou bien une solution eau ethyl ne glycol dans une proportion de 50 50 dans l changeur de chaleur Ne jamais faire circuler une solution d antigel dans l changeur de chaleur Ne pas utiliser avec un tampon une temp rature au dessus de 45 C Toutes les pi ces en plastique sont pr vues pour r sister une temp rature constante de 45 e Faire circuler l eau dans l changeur vertical pour minimiser l chauffement afin d viter des dommages irr parables l instrument Ne pas connecter l changeur vertical circu lation d eau un robinet ou quelque source de refroid
11. ct 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 and place it into a tray 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 Q Discard the blotting paper but reuse the sponges Rinse the unit immediately after use See the Care and maintenance section on the next page pll Use lt 20 methanol methyl alcohol in transfer buffers is the only exception epl2 Care and maintenance Cleaning Do not autoclave or heat any part above 45 e Do not expose to alcohols or organic solvents e Never use abrasive detergents e If using radioactive reagents decontaminate the unit with a cleaning agent such as Contrad 70 or Decon 90 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 electrode 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
12. f blotting paper if the stack seems tight replace the top sponge 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 for gels lt 1 5 mm blotting paper gel membrane blotting paper one 3 mm sponge Assemble the cassette in a tray containing transfer buffer about 3 cm deep Install the cassette s If transferring only one or two gels choose the cassette positions nearest the center The cassettes 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 cassette 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 remove buffer as required so that the level falls between the minimum and maximum buffer level lines Buffer above the maximum buffer level line may cause corrosion of the electrical contacts Note Take care in orienting
13. gel before transfer Use a thinner gel Reduce the gel acrylamide concentration Check that the buffer pH is close to the intended pH Most buffers 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 p13 problem solution Diffuse band patterns Inefficient binding to membrane Chemical parameters Membrane parameters e pl4 Transfer immediately after electrophoretic separation If equilibrat ing before the transfer shorten or eliminate the equilibration time or move the gel to the cold room during equilibration f transfer buffer contains methanol 210 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
14. he appropri ate transfer buffer Chill the buffer before use if possible Prepare the unit Rinse the transfer tank and cassettes with distilled water Active cooling is optional but strongly recommended If no active cooling will be used go to step 3 Note Connect the heat exchanger to a circulator bath such as the MultiTemp III 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 circulat ing only water If using 50 50 ethylene 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 3 8 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 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 especiall
15. 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 circulator bath using water should be set to about 10 C If using 50 50 ethyl ene glycol water the temperature can be set lower Chill the buffer before use if possible Typical transfer parameters Parameters for your sample and buffer system must be determined empirically protein nucleic acids Buffer Towbin 1X TBE or 1X TAE Current A 0 4 0 3 Voltage V 100 50 Transfer time 1 hour 1 hour Coolant temp 10 C 10 or less Note It is a good idea to stain the gel to determine the completeness of the transfer Note Do not store used buffer with transfer tank Chill buffer to 10 C before reuse 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 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 After the transfer is complete Turn the voltage and current settings to zero and turn off the power supply Disconne
16. isse ment dont la pression n est pas r guli re e Pour des coulages plus long on peut aussi contr ler la temp rature en refroidissant le tampon avant l utilisation et ou en utilisant l instrument dans une chambre froide e 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 Ne jamais exposer l appareil des alcools et des solvents organiques Les alcools et les solvents organiques causent des dommages irr parables l appareil e 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 rendues in fficaces Seulement les accessoires et pi ces detach es approuv s ou fournis par Hoefer Inc sont recommand s pour l utilisation l entretien et r paration de cet appareil L utilisation de m thanol une concentration finale de 20 dans les tampons de transfert constitue la seule exception p5 Note Refer to the Electrotransfer Notes section for a discussion of membranes and buffers Note For quick and easy connec tions install Quick fit coupler fittings with valves in the line Operating instructions Perform the transfer as soon as possible after electrophoresis to minimize band diffusion Each step is described below Prepare the buffer Prepare a minimum of 1 5 liters of t
17. it 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 conditions de livraison n est valable que si le produit a t install et utilis conform ment aux instructions ournies par Hoefer Inc La soci t Hoefer Inc ne sera en aucun cas responsable de out dommage caus directement ou indirectement par toute utilisation incorrecte ou non approuv e du produit ou d cou ant 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 limita ion quant la nature de ces dommages piii Deutsch O piv gt Wichtige benutzerinformationen F r ein vollstandiges Verst ndnis und eine sichere Hand habung dieses Produktes ist 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 Doku mentation hinweisen 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 Hoefer Inc 953 Indiana Street San Francisco
18. l levels cassette hook De heat exchanger ports 2 Included but not shown Gel cassettes 4 Foam sponges 6 mm thick 4 e Foam sponges mm thick 8 e Blotting paper sheets 25 heat exchanger pressure safety valve English A A Important information e The safety lid must be in place before connecting the power leads to a power supply e Turn all power supply controls off and disconnect the power leads before removing the safety lid e Rinse only the electrodes not the banana plugs with distilled water before use e Circulate only water or 50 50 water ethylene glycol through the heat exchanger Never use anti freeze mixtures in the heat exchanger Do not operate with buffer temperature above 45 C All plastic parts are rated for 45 C continuous duty e 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 somewhat by chill ing 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 overstuffing the cassette Excess materials may result in cassette damage Never expose any part of the instrument to
19. ly tested to ensure that it meets ts published specifi cations The warranty included in the conditions of delivery is valid only if the product has been installed and used accord ing to the instructions supplied by Hoefer Inc Hoefer Inc shall in no event be liable for incidental or conse quential 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 Francais A 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 instructions importantes 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 exposition la haute tension Tous vos commentaires sur ce manuel seront les bienvenus et veuillez les adresser Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc se r serve le droit d effectuer des modifications de ces sp cifications sans aucun pr avis Garantie et responsabilit Hoefer Inc garantit l utilisateur que le produ
20. mall molecu lar species for instance migrate quickly but often do not bind as well as larger molecules 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 orientation 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 Condi tions 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 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 comprehen sive 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 membrane If this step is skipped band distortion or loss of resolution could result Instrument guidelines Cooling Considerable Joule heat is generated during any transfer because
21. manual TE 22 Hoefer TE 22 tank transfer unit um TE22 IM Rev E0 08 04 H O e fe F Page finder Transfer Electrophoresis Unit function and description 1 Specifications socio 2 Important information 4 Operating instructions 6 and maintenance 12 Troubleshooting Seve cde a dada tu es 13 Electrotransfer notes 15 Bibliography and references 22 Ordering information 24 English O pii A A Safety warnings and precautions 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 important operating and maintenance instructions in the literature accompanying 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 at Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA support hoeferinc com Hoefer Inc reserves the right to make changes in the specifi cations without prior notice Warranty and liability Hoefer Inc guarantees that the product delivered has been thorough
22. n 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 anti sera a method for studying antibody specificity and structure Proc Natl Acad Sci USA 76 3116 1979 Sambrook J and Russell D W Molecular Cloning A Laboratory Manual Cold Spring Harbor Labora tory Press A1 17 2001 Southern E M Detection of specific sequences among DNA fragments separated by gel electrophoresis J 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 Immu nological detection of proteins after electropho retic 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 Electro phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications Proc Natl Acad Sci USA 76 4350 4354 1979 e p23 Ordering information product quantity code no Hoefer TE 22 Tank Transfer Unit 1 TE22 Includes 4 gel cassettes 8 foam sponges 3 mm thick 4 foam sponges 6 mm thick 25 sheets of blotter paper Accessories and replacement parts Gel cassette 2 foam sponges
23. ncing 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 08 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 discussion 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 4 44 g 3 cyclohexylamino 1 propanesulfonic acid Dissolve in 1 5 liters distilled water adjust to pH 11 0 with conc NaOH Adjust volume to 2 0 liters e p19 e p20 Nucleic acid transfers Nucleic acids must normally be transferred in denatured form for most efficient binding RNA is normally denatured with glyoxal before sepa ration or separated in denaturing gels contain ing formaldehyde or methyl mercury
24. ns Vol 1 Section 7 CRC Press 1988 Burnette W N Western blotting electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated 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 and Palade Protein blotting in uniform or gradient electric fields Anal Biochem 144 32 40 1985 Gershoni J M and Palade G E Electrophoretic trans fer of proteins from sodium dodecyl sulfate poly acrylamide gels to a positively charged membrane filter Anal Biochem 124 396 405 1982 Gershoni J M and Palade G E Protein Blotting Principles and Applications Anal Biochem 131 1 15 1983 Gibson W Protease facilitated transfer of high molec ular weight proteins during electrotransfer to nitro cellulose Anal Biochem 118 1 1981 e p22 Lin W and Kasamatsu H On the electrotrans fer of polypeptides from gels to nitrocellulose membranes Anal Biochem 128 302 311 1983 Matsudaira P Sequence from Picomole Quantities of Proteins Electroblotted onto Polyvinylidene Difluo ride Membranes J Biol Chem 262 10035 1987 Ohmsted J B Affinity purificatio
25. nt binding to nitro cellulose 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 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 stain ing background tends to be very high Properly quenched however this paper can be used for antibody detection or other overlay methods of protein 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 trans fer of protein from an SDS gel Burnette 1981 and Symington et al 1981 investigated the effect of the molecular weight of protein 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 Tris cannot be used as in peptide seque
26. of the high current employed so active cooling is recommended especially for transfers requiring 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 condi tions 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 dangerous 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 e p17 e p18 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 efficie
27. rived If any part is missing contact Hoefer Inc Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit This declaration of conformity is only valid for the instrument when it is e used in laboratory loca tions e used as delivered from Hoefer Inc except for alterations described in the User Manual and e connected to other CE labeled instruments or products recommended or approved by Hoefer Inc 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 up to four 9 x 10 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 14 x 24 x 16 5 cm 5 5 x 9 5 x 6 5 in EN61010 1 UL3101 1 CSA C22 2 1010 1 CE Fig 1 Tank transfer unit main components color coded leads A power supply capable of deliver ing up to 100 V and 400 to 500 mA is required WN cover x transfer tank up to four cassettes fit into q the slots S gt electrode panels 2 electrode retaining screw 2 A eg fi
28. t pH Dilute to 1X before use to yield 90 mM Tris 90 mM boric acid and 2 mM EDTA This dilution is commonly 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 commonly 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 Current Protocols in Molecular Biology 1993 A 2 1 PSambrook J and Russell D W 2001 Molecular Cloning A Labora tory Manual A1 17 e p21 E Bibliography and references Alwine J C Kemp D J and Stark G R Method for detection of specific RNAs in agarose gels by trans fer 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 Electropho retic transfer of proteins and nucleic acids from slab gels to diazobenzyloxymethyl cellulose or nitrocellulose sheets Anal Biochem 102 459 471 1980 Bjerrum O J Larsen K and Heegaard N CRC Handbook of Immunoblotting of Protei
29. 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 metha nol buffer system independent of the presence of SDS and under most conditions nucleic acids are negatively charged and also migrate toward the anode Important Never allow the buffer temperature to exceed 45 C Excessive heat will cause the unit to warp e plo Final assembly and transfer 0 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 Use only an approved power supply such as the Hoefer PS 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 and the black lead into the black output jack In most systems the red lead
30. y between the membrane and gel blocks transfer ends of each length of tubing to the circulator bath ports one to the inlet and the other to the outlet Secure 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 approxi mately 0 7 liters Set the stirrer 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 Assemble the transfer cassette Pre wet nitrocellulose or nylon membranes with distilled water Pre wet PVDF or 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 trans fer 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
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