TE22 User Manual – English
Contents
1. Use lt 20 methanol methyl alcohol in transfer buffers is the only exception e plo Care and maintenance Cleaning e Do not autoclave or heat any part above 45 C Do not expose to alcohols or organic solvents Never use abrasive detergents f 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 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 Remove all trapped air pockets in the transfer stack assembly the membrane 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 Us2. v line suunnitellaan sis laboratorio k yt lle vain Vain lis varusteet ja osat hyv ksyiv t tai toimitti Hoefer Inc oheen voi k ytt k ytt miselle valvoalle ja servicing t m tuote Vain k ytt k ytt j nnitett joka on CE merkitsi tai turvallisuus joka on todistanut aidoksi ohi joka on kansallisesti tunnustettnut testaaminen labo ratoriota Turvallisuuskansi t ytyy olla paikallaan ennen yhdist minen k ytt j nnitelyijyj k ytt j nnit teeseen Kiert kaikki k yttoj nnitevalvonnat ja irrottaa valtalyijyt ennen poistaminen turvallisuuskantta Kiert vain vesi tai 50 50 vesi ethylene glycol siin tapauksessa varustetun l mm nvaihtimen l pi l yhdist l mm nvaihdinta vesinapautuk seen eik j hdytysnestel hteeseen miss vesi paine on unregulated Pakkasneste eik orgaaninen liuotin v lineen osassa ei esitele Koskaan Orgaaniset liuottimet aiheuttavat korvaamattoman vahingon yksikk n Ei k yt puskuria yll olevia l mp tiloja enint n m ritetyill teknisill t smennyksill Ylikuumen eminen aiheuttaa korvaamattoman vahingon yksikk n Information Importante French Si cet quipement est utilis dans une mani re pas sp cifi par Hoefer Inc la protection fourni par l quipement pourrait tre diminu e Cet instrument est concu pour l usage de labora toire int rieur seulement Seulement les accessoires et
3. ch nad maxim ln stanovenou technick mi specifi kacemi P eh t zp sob nenapraviteln po kozen jednotka lt igtig Information Danish Hvis dette udstyr bruges i en m de ikke specifice ret ved Hoefer Inc den beskyttelse som er blevet forsynet af udstyret kan m ske sv kkes Dette instrument er designet for indend rs labora toriumbrug bare Bare tilbeh r og del godkendede eller forsynede ved Hoefer Inc kan m ske bruges for drive funk tionsfejl og betjening dette produkt bruger Bare en stramforsyning der er CE markerede eller sikkerhed som er blevet attesteret af en som nationalt er blevet anerkendt prove laboratorium Sikkerhedl get m veere p plads fer forbinding stramforsyningsblyet til en str mforsyning Drejer alle stramforsyningskontroller af og afbryder kraftblyet for fjerning sikkerhedl get Cirkulerer bare vand eller 50 50 vand ethylene glykol gennem varmeveksleren i s fald udrustet Forbind ikke varmeveksleren til en vandhane eller nogen kelemiddelkilde hvor vandtrykket er unregulated Introducerer Aldrig antifreeze eller noget organisk oplesningsmiddel ind i nogen del af instrumentet Organiske opl sningsmidler vil for rsage uboelig skade til enheden Driver ikke med st dpudetemperaturer over maksimummet specificerede tekniske specifica tions Overheding vil for rsage uboelig skade til enheden e pii Belangrijke Informatie Dutch Indien deze u
4. 1 508 893 8999 Fax 1 508 893 0176 E mail support hoeferinc com Web www hoeferinc com Hoefer is a registered trademark of Hoefer Inc Contrad 70 and Decon 90 are trademarks of Decon Lab O 2012 Hoefer Inc All rights reserved Printed in the USA Hoefer
5. Teile genehmigten oder lieferten durch Hoefer Inc kann f r das Funktionieren das Aufrechterhalten und die Wartung dieses Produk tes verwendet werden Verwenden Sie nur eine Energieversorgung die CE gekennzeichnet oder durch ein national anerkanntes Probelaboratorium bescheinigte Sicherheit ist DerSicherheitsdeckel muss im Platz vor dem AnschlieBen der Energieversorgung sein f hrt zu einer Energieversorgung Alle Energieversorgungssteuerungen abdrehen und die Macht trennen f hrt vor dem Entfernen des Sicherheitsdeckels Nur Wasser oder 50 50 Glykol des Wassers Athylens durch den W rmeaustauscher wenn so ausgestattet in Umlauf setzen Verbinden Sie den W rmeaustauscher mit einem Wasserklaps oder jeder K hlmittel Quelle nicht wo der Wasserdruck ungeregelt wird F hren Sie nie Frostschutzmittel oder jedes organische L sungsmittel in jeden Teil des Instru mentes ein Organische L sungsmittel werden nicht wiedergutzumachenden Schaden der Einheit verursachen Mit Puffertemperaturen ber angegebenen technischen Spezifizierungen des Maximums nicht funktionieren Die berhitzung wird nicht wiedergutzumachenden Schaden der Einheit verursachen Informazioni Importanti Italian Se quest apparecchiatura usata in un modo specificato da Hoefer Inc la protezione fornito dall apparecchiatura potrebbe essere indebolita Questo strumento disegnato per l uso di labora torio interno solo
6. V and 400 to 500 mA is required J NS cover NS transfer tank up to four cassettes fit into q the slots OS electrode panels 2 electrode retaining screw 2 aq SCH fill levels cassette hook K 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 ports 2 heat exchanger pressure safety valve 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 the appropri ate transfer buffer Chill the buffer before use if possible Prepare the unit o Rinse the transfer tank and cassettes with distilled water e 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 RCB20 PLUS 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 O 7 bar 10 psi above atmospheric pressure Set the temperature to 10 C or higher if circulat ing
7. 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 p17 e p18 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 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 fragm
8. estoque de poder leva a um estoque de poder Desliga todos controlos de estoque de poder e desconecta os chumbos de poder antes de retirar a tampa de seguran a Circulam s gua ou 50 50 glicol de gua ethylene pelo exchanger de calor se for assim equiparam N o ligue o exchanger de calor a uma torneira de gua nem qualquer fonte de refrigerante onde a press o de gua n o regulado Nunca introduz anticongelante nem qualquer org nico solvente em qualquer parte do instru mento Org nico solvente causar agress o irrepar vel unidade N o opera com temperaturas de buffer acima do m ximo especificou especifica es t cnicas Super aquecer causar agress o irrepar vel unidade Informaci n Importante Spanish Si este equipo es utilizado en una manera no espe cificado por Hoefer Inc la protecci n proporcio nado por el equipo puede ser da ada Este instrumento es dise ado para el uso interior del laboratorio s lo S lo accesorios y partes aprobaron o suministraron por Hoefer Inc puede ser utilizado para operar para mantener y para atender a este producto S lo utiliza una alimentaci n que es CE marc o la seguridad certificada por un nacionalmente reconocido probando el laboratorio Latapa de la seguridad debe estar en el lugar antes de conectar la alimentaci n lleva a una alimentaci n Apaga todos controles de alimentaci n y desco necta los plomos del poder ante
9. 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 arrived 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 locations e used as delivered from Hoefer Inc except for alterations described in the User Manual and 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 Dimensions wxh xd Product certifications up to four 9x 10 cm gels 5O 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 Installation category II Pollution degree 2 14x24x16 5 cm 5 5x9 5x6 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 delivering up to 100
10. 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 e p13 e pl4 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 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 optim
11. 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 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 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 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 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 e 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 a
12. Qu Hoefer TE22 Tank transfer unit um TE22 IM Rev GO 07 12 H O e fe r B Contents Important Informatica ii Waste Electrical and Electronic Equipment WEE vii Transfer Electrophoresis Unit function and description seesssesssse 1 SPECIFICATIONS ERE 2 Operating IRStUCBOTS rip NA 4 Care and maintenance nenne 10 Tro bleshooting 2 inire co nnne rion V Electrotranster notes usus etre hni 13 Bibliography sess ses meer usa rear acia 20 Ordering Information 22 Important Information English If this equipment is used in a manner not speci fied by Hoefer Inc the protection provided by the equipment may be impaired This instrument is designed for indoor laboratory use only Only accessories and parts approved or supplied by Hoefer Inc may be used for operating main taining and servicing this product Only use a power supply that is CE marked or safety certified by a nationally recognized test ing laboratory The safety lid must be in place before connecting the power supply leads to a power supply Turn all power supply controls off and disconnect the power leads before removing the safety lid Circulate only water or 50 50 water ethylene glycol through the heat exchanger if so equipped Do not connect the heat exchanger to a water tap or any coolant source where the water pressure is unregulated Never introduce antifreeze or any organic sol
13. Solo gli accessori e le parti hanno approvato o hanno fornito da Hoefer Inc potrebbe essere usato per operare per mantenere e per revisionare questo prodotto usa Solo un alimentatore che CE ha marcato o la sicurezza certificato da un nazionalmente ricon osciuto testando il laboratorio Il coperchio di sicurezza deve essere nel luogo prima di collegare i piombi di alimentatore a un alimentatore Spegne tutto i controlli di alimentatore e disin serisce i piombi di potere prima di togliere il coper chio di sicurezza Circola solo l acqua o 50 50 glicole di acqua etilene attraverso lo scambiatore di calore se cosi equipag giato Non collegare lo scambiatore di calore a un rubinetto di acqua o qualunque fonte di refriger ante dove la pressione di acqua sregolata Non introduce mai l antigelo o qualunque solvente organico in qualunque parte dello strumento solventi organici causeranno il danno irreparabile all unit Non opera con le temperature di tampone al di sopra del massimo ha specificato le descrizioni tecniche II surriscaldamento causera il danno irreparabile all unit Viktig Informasjon Norwegian Hvis dette utstyret blir brukt i en m te ikke spesi fisert ved Hoefer Inc beskyttelsen som ha blitt git av utstyret kan bli svekket Dette instrumentet er utformet for innenders labo ratoriumbruk bare Bare tilbehor og deler godkjente eller forsynte ved Hoefer Inc kan bli brukt fo
14. e Important Never allow the buffer temperature to exceed 45 C Excessive heat will cause the unit to warp Final assembly and transfer o 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 gt or black lead Make sure the banana plugs seat into the connectors in the lid e Use only an approved power supplies such as the Hoefer PS2A200 PS200HC or PS300B 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 is the anode and the black lead is the cathode e 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 ethylene 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 ti
15. e 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 causes it to bow see the recommended assembly instructions on page 6 Grid pattern on membrane 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 Increase the field strength migrate out of gel 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 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 6 7 increases the positive charge on proteins higher pH 26 7 increases the negative charge on proteins e pl
16. embrane 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 e p12 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 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
17. en Beauftragten des Herstellers auf um Informationen hinsichtlich der Entsorgung Ihres Ger tes zu erhalten Questo simbolo indica che i rifiuti derivanti da apparecchiature elettriche ed elettroniche non devono essere smaltiti come rifiuti municipali indifferenziati e devono invece essere raccolti separatamente Per informazioni relative alle modalit di smantellamento delle apparecchiature fuori uso contattare un rappresentante autorizzato del fabbricante Este s mbolo indica que el equipo el ctrico y electr nico no debe tirarse con los desechos dom sticos y debe tratarse por separado Contacte con el representante local del fabricante para obtener m s informaci n sobre la forma de desechar el quipo Denna symbol anger att elektriska och elektroniska utrustningar inte f r avyttras som osorterat hush llsavfall och m ste samlas in separat Var god kontakta en auktoriserad tillverkarrepresentant f r information ang ende avyttring av utrustningen e pvii 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
18. ents 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 M 18 6 g Dissolve in 7O 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 0g 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 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 nec
19. essary 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 Laboratory Manual A1 17 e p19 A Bibliography 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 Proteins 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 KE 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 Electrophor
20. etic 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 p20 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 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 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 pape
21. ide grey bottom anode side ZZ AS ZS SS O Y A A P A O IO E A4 N ZS E M E ZS Z AS EAA SS O ee E ZS i A NO M E A M M A4 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 o Close the cassette and press lightly to lock the tabs The assembled cassette should hold the gel in firm contact with the membrane without sgueezing the gel If the stack seems loose add sheets of 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 o If transferring only one or two gels choose the cassette positions nearest the center The cas
22. itrusting in een manier wordt gebruikt die niet door Hoefer Inc is gespecificeerd de bescherming die door de uitrusting is verzorgd kan worden geschaad Dit instrument is voor binnenlaboratoriumgebruik enkel ontworpen Enkel onderdelen en delen keurden goed of leverden door Hoefer Inc kan voor het bedienen worden gebruikt handhavend en onderhouden van dit product gebruik Enkel een netvoeding die CE is markeerde of veiligheid die door een is gecertificeerd die nationaal is herkend testene laboratorium Het veiligheidsdeksel moet in plaats voor het verbinden van de netvoeding leidt tot een netvoeding zijn Doe alle netvoedingscontroles Uit en koppel los de machtleiding voor het verwijderen van het veiligheidsdeksel Circuleer enkel water of 50 50 water ethyleen glycol door de hitte exchanger zo ja uitrust Verbind de hitte exchanger naar een waterkraan of koelmiddelbron niet waar de waterdruk niet geregulariseerd is Stel Nooit antivriesmiddel of organische oplosmid delen in deel van het instrument voor Organische oplosmiddelen zullen onherstelbare schade aan de eenheid veroorzaken Bedien niet met buffertemperaturen boven het maximum specificeerde technische specificaties Oververhittend zal onherstelbare schade aan de eenheid veroorzaken T rke Tietoa Finnish Jos tata varusteita k ytet n tavassa ei m ritetty Hoefer Inc suojelu ehk isty varusteille saattaa olla avuton T m
23. l problem solution Diffuse band patterns 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 If 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 that the preferred binding surface of the membrane if any contacts the gel Inefficient binding to membrane Chemical parameters 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 Membrane parameters 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 m
24. les parties ont approuv ou ont fourni par Hoefer Inc pourrait tre utilis pour fonctionner maintenir et entrete nir ce produit utilise Seulement une alimentation qui est CET a marqu ou la s curit certifi par un nationale ment reconnu essayant le laboratoire Le couvercle de s curit doit tre sa place avant connecter l alimentation mene une alimentation Tourner tous contr les d alimentation de et d brancher les avances de pouvoir avant enlever le couvercle de s curit Circuler seulement de l eau ou 50 50 glycol d eau thyl ne par l exchanger de chaleur si si quip Ne pas connecter l exchanger de chaleur un robinet d eau ou la source d agent de refroidissement o la pression d eau est non r gul e Ne Jamais introduire d antigel ou du dissolvant organique dans n importe quelle partie de e piii l instrument Les dissolvants organiques causeront des dommages irr parables l unit Ne pas fonctionner avec les temp ratures de tampon au dessus du maximum a sp cifi des sp cifications techniques La surchauffe causera des dommages irr parables l unit Wichtige Informationen German Wenn diese Ausr stung gewisserma en nicht angegeben durch Hoefer Inc verwendet wird kann der durch die Ausr stung zur Verf gung gestellte Schutz verschlechtert werden Dieses Instrument wird f r den Innenlaborge brauch nur daf r entworfen Nur Zus tze und
25. me 1 hour 1 hour Coolant temp 10 C 10 C or less o 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 e 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 o Turn the voltage and current settings to zero and turn off the power supply Disconnect the leads from the power supply jacks e 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 e 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 on the next page
26. pproxi mately O 7 liters o Set the stirrer to low medium which accomplishes buffer circulation without forcing buffer through the cassettes Assemble the transfer cassette o 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 e 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 e 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 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 s
27. przez uznane na poziomie krajowym laborato rium badawcze Bezpiecze stwo lid musi by w miejsce przed pod czeniem zasilania prowadzi do zasilania Za wszystkie r d a zasilania urz dzenia steruj ce off i od czy moc prowadzi przed odbiorem bezpiecze stwa lid Kr tylko wody lub wody 50 50 ethylene glycol wymiennik ciep a poprzez je li tak wyposa one Nie nale y po czy wymiennik ciep a woda z kranu lub jakimkolwiek ch odziwo r d a je eli ci nienie wody jest nieuregulowanych Nigdy nie wprowadza rozpuszczalnika organ icznego przeciw zamarzaniu lub jakichkolwiek na dowoln cz dokumentu Rozpuszczalniki organiczne spowoduje nieodwracalne szkody dla jednostki Nie dzia aj w buforze temperatury powy ej maksymalnego okre lone specyfikacje techniczne Przegrzania spowoduje nieodwracalne szkody dla jednostki Informa es Importantes Portuguese Se este equipamento usado numa maneira n o especificada por Hoefer Inc que a protec o forne cida pelo equipamento pode ser comprometida Este instrumento projectado para uso de interior de laborat rio s S acess rios e partes aprovaram ou forneceu por Hoefer Inc pode ser usada para operar manter e servicing este produto S usa um estoque de poder que CE marcou ou seguran a registrada por um nacionalmente recon hecido testando laborat rio A tampa de seguran a deve estar em lugar antes de ligar o
28. r 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 p21 Ordering information product quantity code no Hoefer TE22 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 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 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 9x 10 5 cm 50 TE26 Companion products Hoefer PS2A200 Power Supply 200 V 2A 1 PS2A200 Hoefer PS200HC Power Supply 200 V 2A 1 PS200HC Hoefer PS300B Power Supply 300 V 0 5A 1 PS300B e p22 Hoefer Inc 84 October Hill Road Holliston MA 01746 Toll Free 1 800 227 4750 Phone
29. r drive vedlikeholde og betjene dette produktet bruker Bare en kraftforsyning som er CE merket eller sikkerhet som ha blitt sertifisert av et som nasjonalt ha blitt anerkjent prover laboratorium e piv Sikkerheten lokket m veere p plass far forbinding kraftforsyningene blyene til en kraftforsyning Vender all kraftforsyningsstyring av og frakopler kreftene blyene far fjerning sikkerheten lokket Sirkulerer bare vann eller 50 50 vann ethylene glykol gjennom oppvarmingen veksleren i s fall utstyrer Ikke forbind oppvarmingen veksleren til en vanntapp eller noe kjalemiddelkilde hvor vannet trykket er unregulated Introduserer Aldri antifreeze eller noe organisk losemiddel inn i noe del av instrumentet Organi ske losemiddler vil for rsake irreparabel skade p enheten Driver med buffertemperaturer over maksimum ikke spesifiserte teknisk spesifikasjoner overop pheting vil for rsake irreparabel skade p enheten Wazne Informacje Polish Jezeli ten sprzet jest wykorzystywany w spos b nie okreslone przez Hoefer Inc do ochrony przewid zianej przez urzadzenie moze zostac obnizony Instrument ten jest przeznaczony do uzytku w laboratoriach kryty tylko Tylko akcesori w i czesci zatwierdzone lub dostarc zone przez Hoefer Inc mog by wykorzystane do eksploatacji utrzymania i obstugi tego produktu korzysta jedynie zasilacza ze jest nosz ce ozna kowanie CE lub bezpiecze stwa uwierzytelnione
30. rustad fall Inte kopplar v rmen exchanger till en vatten kran eller n got kylmedel k lla d r vattnet trycket r unregulated Inf r aldrig kylv tska eller n got organiska l sningsmedel in i n gon del av instrumentet Organiskt l sningsmedel ska orsaka irreparable skada till enheten Anv nd inte med buffert temperaturer ver det h gsta angivna tekniska specifikationerna verhettning skulle orsaka irreparabla skador p enheten e pvi English Ea French X German X pon Italian M Spanish 134 Swedish yd gum Waste Electrical and Electronic Equipment WEEE This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of your equipment Ce symbole indique que les d chets relatifs l quipement lectrique et lectronique ne doivent pas tre jet s comme les ordures m nag res non tri es et doivent tre collect s s par ment Contactez un repr sentant agr du fabricant pour obtenir des informations sur la mise au rebut de votre quipement Dieses Symbol kennzeichnet elektrische und elektronische Ger te die nicht mit dem gew hnlichen unsortierten Hausm ll entsorgt werden d rfen sondern separat behandelt werden m ssen Bitte nehmen Sie Kontakt mit einem autorisiert
31. s de quitar la tapa de la seguridad Circula s lo agua o 50 50 glicol de agua etileno por el intercambiador de calor si se es el caso equiparon No conecte el intercambiador de calor a un toque de la agua ni cualquier fuente del l quido refrigerante donde la presi n del agua est libre Nunca introduce anticongelante ni alg n solvente org nico en cualquier parte del instrumento Los solventes org nicos causar n da o irreparable a la unidad No opera con temperaturas de b fer encima del m ximo especific especificaciones t cnicas Reca lentar causar da o irreparable a la unidad Viktig Information Swedish om denna utrustning anv nds i ett s tt som inte har specificeras av Hoefer Inc skyddet tillhan dah ll vid utrustningen kan skadas Detta instrument formges f r inomhuslaborato rium anv ndning bara Bara medhj lpare och delar godk nde eller lever erade vid Hoefer Inc kan anv ndas f r fungera underh lla och servicing denna produkt anv nder bara en kraft tillg ng som r CE markerade eller s kerhet intygade vid en nationellt erk nd testande laboratorium S kerheten locket m ste vara p platsen f re koppla kraften tillg ngen blyen till en kraft tillg ng Vander sig alla kraft tillg ng kontroller av och kopplar bort kraften blyen f re flytta s ker heten locket Cirkulerar bara vatten eller 50 50 vatten ethylene glycol genom v rmen exchanger i s ut
32. 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 cassette at a time and slide it into a set of vertical slots Do not discard the buffer e 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 e 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 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 anod
33. tibody detection or other overlay methods of protein identification A summary of membrane type and recommended methanol concentration follows Membrane type Methanol 6 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 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 8g SDS FW 288 4 0 196 3 5 mM 2 08 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
34. um 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 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 250 mA Po
35. vent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not operate with buffer temperatures above the maximum specified technical specifications Overheating will cause irreparable damage to the unit Dulezit Informace Czech Pokud by toto za zen je pou ito zp sobem kter nen podle Hoefer Inc ochrana poskytovan na z klad za zen m e b t naru ena Tento n stroj je ur en pro vnit n pou it v laborato i pouze Pouze p slu enstv a sti schv len nebo poskyt nut ch Hoefer Inc mohou b t pou ity pro provoz dr bu a dr b tohoto v robku zdroj nap jen pou vaj jen e je opat en ozna en m CE osv d ena nebo bezpe nost vnitrost tn uznan mi zku ebn mi laborato Bezpe nosti lid mus b t zavedena p ed p ipojen m nap jec zdroj nap jen vede k Turn ve ker nap jen kontroly vypnuto a odpojit p ed odb rem energie vede bezpe nostn v ko Rozeslat pouze voda nebo 50 50 voda ethyleng lykolu prost ednictv m v m n k tepla je li to vybav ena Nemaj p ipojen v m n k tepla s vodn mi set epn nebo jak koli chladic kapaliny zdroje kde tlak vody je neregulo Nikdy zav st prost edek proti zamrznut nebo jak koli organick rozpou t dla do jak koli sti z tohoto n stroje Rozpustidl m zp sob nenapravi teln po kozen jednotka Nejsou provozov na s pufru teplot
36. wer 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 p15 e pl6 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 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 an
Download Pdf Manuals
Related Search