Home

miniVE - GE Healthcare Life Sciences

1. d Lay the membrane on the gel Do not reposition the membrane once it contacts the gel e Lay one piece of wet filter paper on the membrane f Lay two packing sponges on the filter paper A second transfer stack if added is placed between these two sponges Repeat steps b e 19 Note Avoid wetting the banana plugs with electrophoresis buffer A protective film of GelSealTM supplied can be applied to the plugs as a corrosion barrier Figure 3 3 Final assembly 20 Check the position of the transfer stack It should be centered on the elec trode plate no layer should be pinched when the module is closed Fold the empty half of the cup over the stack and press the halves together to snap the module closed 3 4 Final assembly 1 D Color coded leads connect the electrodes to the power supply Banana plug connectofs 2 per module Pour 300 350 ml of transfer buffer into the top of the module Tap the blot ting cup lightly to dislodge any air bubbles in the packing sponges Position the module s in the tank with the banana plugs toward the center the red side facing outward Add deionized water to the tank 1 7 liters for one module and 1 2 liters for two modules Buffer temperature should not exceed 75 C to avoid rapid evaporation Passive cooling is recommended if the transfer will be longer than one hour if biological activity must be protected or if transferring nucleic acids Chill dei
2. Amersham Biosciences beh lt sich das Recht vor die Spezifikationen ohne vorhergehende Ank ndigung zu ndern 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 cifica tions et normes en vigueur La garantie incluse dans les conditions de livraison n est valable que si le pro duit a t install et utilis conform ment aux instruc tions fournies par Amersham Biosciences La soci t Amersham Biosciences ne sera en aucun cas responsable de tout dommage caus directement ou indirectement par toute utilisation incorrecte ou non approuv e du produit ou d coulant de cette utilisation y compris toute perte de b n fice ou de recettes toute perte de perspectives commer ciales 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 1998 Amersham Biosciences Tous droits r serv s La reproduction le stockage dans un syst me de r cup ration d informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la pr sente publication en totalit ou en partie sont strictement interdits sans autorisation pr alable crite de la soci t Gew hrleistung and Haftung Amersham Biosciences garantiert daf das gelieferte Produkt sorgf
3. Dialyze or desalt the sample V Centrifuge or filter the sample to remove particulates 15 Poor band resolution Y Y Y Y Y Use only the highest quality reagents Only use freshly deionized urea Use only gels that were recently prepared Check pH values of the resolving and stacking gel solutions Do not back titrate buffers Conduct the separation at a lower current or voltage setting Sample preparation Y Y Y Y Dialyze or desalt the sample Reduce the sample volume or concentration Improve dissociation of protein subunits by heating sample in SDS sample buffer 1 2 minutes at 100 C Store on ice after heating Store sample on ice before it is denatured Add protease inhibitors if necessary to prevent proteolytic degradation of sample Add more mercaptoethanol or dithiothreitol check sample treat ment Store samples to be frozen in aliquots to prevent repeated thaw ing Store at 40 C to 80 C Protein streaks vertically Y Y Centrifuge or filter the sample to remove particulates Dialyze or desalt the sample Bromophenol blue doesn t sharpen into a concentrated zone in the stacking gel Y Y 16 Pour a taller stacking gel For best results allow a stacking gel height of 2 5 times the height of the sample in the well Dispose of outdated acrylamide solutions and use only the highest grade of acrylamide When preparing samples avoid using solutions with a high sodium
4. 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 installiert und benutzt wurde Amersham Biosciences bernimmt keinerlei Haftung f r Sch den oder Folgesch den einschlie lich aber nicht begrenzt auf Gewinneinbufen Einkommensverluste entgangene Gesch ftsabschl sse Verlust der Gebrauchsf hig keit oder andere Verluste die wie auch immer durch eine fehlerhafte oder unsachgem e Verwendung des Produkts verursacht wurden Copyright 1998 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 Italiano Espa ol Informazioni importanti per l operatore Per un utilizzo sicuro del prodotto leggere attenta mente l intero contenuto del presente manuale Il punto esclamativo all interno di un tri angolo equilatero indica all operatore la presenza di importanti istruzioni di fun zionamento e manutenzione nella docu mentazione 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
5. tach es p Ne pas enlever les visses des pinces pour ouvrir les pinces il suffit de tourner quatre fois dans le sens inverse des aiguilles d une montre p 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 p Seulement les accessoires et pi ces detach es approuv s ou fournis par Amersham Biosciences sont recommand s pour l u tilisation l entretien et r paration de cet appareil 1 3 Unpacking 1 Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears damaged contact the carrier immedi ately Be sure to keep all packing material for damage claims or for repack ing should it become necessary to return the unit Prior to use wash the tank and module with a dilute solution of non abra sive laboratory detergent Thoroughly rinse first with water and then with distilled water Section Figure 2 1 Module in the closed position Figure 2 2 Module in the open position Note The sealing plate has three positions closed sealed for casting half open for elec trophoresis and fully open for placing the gel sandwich 2 The Electrophoresis Module The Electr
6. ECL Western blotting reagents sufficient for 2000 cm of membrane 1 ECL Western blotting reagents sufficient for 4000 cm of membrane 1 ECL Western blotting system kit sufficient for 1000 cm of membrane Hybond C Super supported pure nitrocellulose 12 x 10 cm 20 pk Hybond C nitrocellulose membrane 12 x 10 cm 20 pk Hybond C pure nitrocellulose membrane 20 x 20 cm 10 pk Hybond C pure nitrocellulose membrane 20 cm x 3 m roll Hybond C pure nitrocellulose membrane 30 cm x 3 m roll Hybond P PVDF membrane 20 x 20 cm 10 pk Hybond P PVDF membrane 30 cm x 3 m roll Hybond ECL nitrocellulose membrane 20 x 20 cm 10 pk Hybond ECL nitrocellulose membrane 6 x 8 cm 50 pk Hybond ECL nitrocellulose membrane 30 cm x 3 m roll Hyperfilm ECL 12 5 x 17 5 cm 25 pk Blotter paper 7 x 8 cm 25 pk Blotter paper 9 x 10 5 cm 50 pk Nitrocellulose 0 45 um pore size 9 x 10 5 cm 10 pk Nitrocellulose 0 45 um pore size 33 cm x 3 m roll Nitrocellulose 0 20 um pore size 33 cm x 3 m roll Nylon 66 Standard 0 45 um pore size 33 cm x 3m roll Nylon Standard GeneBind 0 45 um pore size 20 cm x 3 m roll Nylon 66 Plus charged 0 45 um pore size 33 cm x 3 m roll 2 D Instruments and Accessories IPGphor System 115 230 V Order Strip Holders separately 1 7 cm Strip Holder complete for use 6 pk with Immobiline Drystrip IPG gels Immobiline DryStrip pH 4 7 L 7 cm 12 pk Immobiline DryStrip pH 3 10 L 7 cm 12 pk Immobiline DryStrip pH 3 10 NL 7 cm 1
7. cause shadow bands on the blot Equilibrate the gel in transfer buffer for 10 minutes Equilibration allows the gel to swell or shrink before it contacts the transfer membrane and removes excess buffer salts and detergents from the gel Longer equilibration may result in diffuse bands The transfer membrane is prepared in two steps a Pre wet nitrocellulose or nylon membranes in distilled water taking care not to trap air bubbles dip one end of the membrane into the buffer and slowly submerge allowing it to wet by capillary action Pre wet PVDF or other hydrophobic membranes in methanol b After prewetting soak all membrane types in transfer buffer for 2 5 minutes Wet the two pieces of filter paper in transfer buffer Assemble the transfer stack so that molecules will migrate to the membrane For negatively charged macromolecules such as proteins run in an SDS gel and nucleic acids assemble the transfer stack on the black cathode side Note For best results care should be taken to avoid trapping air bubbles as each layer is applied This is best accomplished by always establishing full contact along one side and maintaining it as the layer is lowered into posi tion See step 5 for proper orientation a Center a packing sponge on the black cathode side b Lay one piece of wet filter paper on the sponge c Position the equilibrated gel on the filter paper Wet the gel surface with a few drops of transfer buffer
8. entering the upper buffer chamber Also verify this Add the appropriate amount of electrophoresis buffer to the upper buffer chamber Fill the upper buffer chamber to a level 3 5 mm above the notched plate For a 10 5 cm long gel approximately 100 ml will be required and for a shorter gel approximately 75 ml 11 5 Prepare and apply the sample Increase liquid sample density with 10 glycerol or sucrose Add a tracking dye such as phenol red or bromophenol blue Underlay the sample into the wells using a micro pipet or fine tipped microsyringe Table 1 shows the volume of sample required for different numbers of wells and comb thicknesses Note The amount of protein sample added to each well depends on both the sensitivity of the staining method and the distribution of protein among separate bands With Coomassie Blue it is possible to detect 1 pg in a single band with the more sensitive silver stains it is possible to detect as little as 10 ng Table 2 1 Volume of sample ul per 1 mm depth Well capacities Comb thickness mm No of wells 0 75 1 0 1 5 5 9 5 12 7 19 1 9 5 8 10 3 6 4 8 7 2 15 2 2 2 9 4 4 18 2 9 6 Electrical connections Figure 2 13 Position the safety lid over the Fully assembled unit and seat the lid so the banana plugs engage the jacks in the lid The lid is symmetrical and fits in either orientation Plug the color coded leads into the jacks of an approved power sup pl
9. inviare eventuali commenti al presente manuale a Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso Informaci n importante para el usuario Para comprender el producto y utilizarlo con seguri dad es necesario leer este manual en su totalidad El signo de admiraci n en un tri ngulo equil tero en el manual advierte al usuario sobre la presencia de instruc ciones importantes de operaci n y man tenimiento del aparato El s mbolo del rayo en un tri ngulo equi l tero alerta al usuario sobre el riesgo de exposici n a altas tensiones Si desearan hacer alg n comentario sobre este manual tengan la amabilidad de remitirlo a Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se reserva el derecho a modificar las especificaciones sin previo aviso Garanzia e responsabilit Amersham Biosciences garantisce che prima della consegna il prodotto stato collaudato a fondo per soddisfare i requisiti specificati La garanzia inclusa nelle condizioni di consegna risulta valida solamente se il prodotto stato installato ed utilizzato nel rispetto delle istruzioni fornite da Amersham Biosciences Amersham Biosciences non potr essere ritenu ta responsabile di incidenti o danni consequenziali inclusi ma non limit
10. polyacrylamide gel electrophoresis J Biol Chem 224 4406 4412 1969 4 2 Blotting 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 and G E Palade Protein Blotting Principles and Applications Anal Biochem 131 1 15 1983 Harlow E and Lane D Antibodies A Laboratory Manual Cold Spring Harbor Press 1988 Sambrook J et al Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press B 23 1989 Towbin H Staehelin T and Gordon J Electrophoretic transfer of proteins from polyacry lamide gels to nitrocellulose sheets procedure and some applications Proc Natl Acad Sci USA 76 4350 4354 1979 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 or would like to arrange to repair it please call or fax your local Amersham Biosciences sales office or representative Important Request a copy of the Health and Safety Declaration Form before returning the item No items can be accepted for servicing or return unless this form is properly completed Ordering Information Qty Code No Hoefer miniVE Basic includes two gel modules lid and tank 1 80 6418 58 Hoe
11. screws should not be tightened to the point that pressure stresses the cassette 6 Check that both gel surfaces will contact buffer Novex gels check that the bottom gel contact slot is exposed 7 Move the sealing plate into the half open position to prepare for elec trophoresis Apply gentle pressure to both tabs and lock them into the lower notch Note Stacking gel resolution is optimal if the gel is poured just before electrophoresis 10 2 1 3 Cast the stacking gel 1 2 ee If the gel has wells skip to Section 2 2 The gel sandwich or cassette should already be in place on the module To ensure seamless contact between the resolving and stacking gels remove residual liquid by blotting one corner of the gel with a lint free tissue Prepare the stacking gel monomer solution Tip To calculate the volume measure the distance in cm from the top of the resolving gel to the notch in the glass plate This should be at least 2 cm Multiply this distance by the gel width 8 3 cm and the gel thickness cm for the required volume ml Deaerate the stacking gel monomer solution add catalyst and initiator and then pour Use a pipette to deliver the solution into one corner of the plate taking care not to trap any bubbles Insert a comb at a slight angle to pre vent trapping air into the sandwich allowing the comb sides to rest on the spacers Allow a minimum of one hour for the gel to polymerize F
12. 2 pk RPN755 RPN756 RPN800 RPN2280 RPN2107 RPN2132 RPN2109 RPN2209 RPN2106 RPN2108 RPN1210G RPN1210C RPN2020W RPN203W RPN303W RPN2020F RPN303F RPN2020D RPN68D RPN303D RPN1674H 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 6414 02 80 6416 11 17 6001 10 17 6001 11 17 6001 12 27 Notes 1 MINIVE ELECTROPHORESIS UNIT FUNCTION AND DESCRIPTION 1 1 SPECIFICATIONS e 1 2 IMPORTANT INFORMATION s s s ss s sn 1 8 UNPACKING a a s Rss 2 THE ELECTROPHORESIS MODULE 2 1 PREPARING THE op 2 2 FINAL ASSEMBLY s 2 3 ELECTROPHORESIS 2 4 AFTER ELECTROPHORESIS 2 5 CARE AND MAINTENANCE 2 6 TROUBLESHOOTING s 3 THE BLOT MODULE 3 1 ASSEMBLY A ll ll s 3 2 PREPARATION a s s s s s s 3 3 PREPARE THE TRANSFER STACK 3 4 FINAL ASSEMBLY s s 3 5 ELECTROTRANSFER n 3 6 AFTER ELECTROTRANSFER 3 7 CARE AND MAINTENANCE 3 8 TROUBLESHOOTING 4 BIBLIOGRAPHY 5 CUSTOMER SERVICE INFORMATION Fran ais Deutsch Renseignements importants d utiliza tion Pour une bonne compr hension et une
13. AMERSHAM BIOSCIENCES mini VE Electrophoresis and Electrotransfer Unit User Manual 80 6420 86 Amersham Rev A 5 98 e Y Biosciences Hoefer miniVE Electrophoresis Unit Function and Description The Hoefer miniVE vertical electrophoresis system performs vertical gel elec trophoresis on mini format gels The basic unit includes two electrophoresis mod ules Each module holds one gel sandwich 10 cm wide and 8 to 10 5 cm long One gel can be cast in place on the electrophoresis module A wide range of accessories ordered separately see Section 5 lends the miniVE a high degree of versatility These include Figure 1 1 Main components Power supply minimum ratings 50 mA 250 V Constant current or constant voltage a large selection of combs and spacers adaptor sleeves to run Novex precast gels and a blot module which converts the miniVE into a mini blotting unit See page 17 for instructions Color coded leads connect the electrodes to the power supply Lid Electrophoresis module Banana plug connectors 2 1 1 Specifications Electrophoresis Gel sandwich size Max tank volume Max voltage Max wattage Electrotransfer Max volume blot module Max tank volume for passive cooling Max wattage Max current miniVE specifications Max operating temperature Chemical compatibility Environmental operating conditions Installation ca
14. Important user information Please read this entire manual to fully understand the safe and effective use of this product The exclamation mark within an equilat eral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the liter ature accompanying the instrument The lightning symbol within an equilat eral 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 Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the 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 includ ing 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 CopyrightO 1998 Amersham Biosciences All rights reserved No part of this public
15. ati a perdite di profitti mancato guadagno perdite di affari difetti di funzionamento e relative esposizioni dovuti ad un utilizzo non corretto del prodotto Copyright 1998 Amersham Biosciences Tutti i diritti riservati Nessuna parte della presente pubblicazione pu essere riprodotta conservata in sis temi di gestione dati o trasmessa in alcun forma n per nessuno scopo senza autorizzazione scritta del produttore Garant a y responsabilidad Amersham Biosciences garantiza que el pro ducto entregado ha sido probado a fondo para com probar el cumplimiento de las especificaciones publi cadas La garant a incluida en las condiciones de entrega s lo es v lida si el producto se ha instalado y utilizado de acuerdo con las instrucciones entregadas por Amersham Biosciences Amersham Biosciences no ser responsable bajo ning n concepto de da os directos o indirectos incluyendo sin limitaci n la p rdida de beneficios la p rdida de ingresos la p rdida de oportunidades de negocio la p rdida de utilizaci n y otras consecuen cias relacionadas cualquiera que sea la causa que se deban a la utilizaci n defectuosa e incorrecta del pro ducto Copyright 1998 Amersham Biosciences Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistema de recu peraci n ni la transmisi n de parte alguna de esta publicaci n sin la autorizaci n por escrito de la empresa English
16. ation may be reproduced stored in a retrieval system or transmitted in any form by any means without permission in written form from the company Printed in the USA
17. d the blotting paper 4 Label each membrane and indicate the sample side Lift the membrane s with blunt forceps and allow to air dry 5 Rinse the unit immediately after use Care and Maintenance D Do not autoclave or heat any part above 75 C D Do not use organic solvents strong or oxidizing cleaning solutions abra sives or strong acids or bases on any part of the instrument Immediately after each use rinse the unit with water and then rinse thoroughly with distilled water Handle the module with care to prevent damage to the elec trode plugs Allow to air dry 21 22 3 8 Troubleshooting Incomplete transfer Blank areas on the membrane Y Y Y Remove all trapped air bubbles in the transfer stack take especial ly great care during stack assembly to prevent air bubbles from forming as each layer is placed Check electrode continuity Use a lower ionic strength buffer Molecules do not migrate out of gel Y i RS SOS Increase the field strength Increase the transfer period Try doubling it Do not expose the gel to staining or fixing agents before transfer Use a thinner gel Reduce the gel acrylamide concentration For proteins use 3 5 mM SDS 0 196 in the transfer buffer Avoid using methanol in the protein transfer buffer or reduce the amount to a minimum Use reagent grade chemicals Increase the length of time Southern blots are depurinated Check the buffer pH Most buf
18. e or one with a smaller pore size 0 10 0 20 um Place a membrane on both sides of the gel if different proteins may be migrating in opposite directions If the sample load may be exceeding the capacity of the binding surface area apply two membranes If blow through occurs reduce the sample load 23 24 Bibliography 4 1 Polyacrylamide gel electrophoresis Adams L D and Gallagher S R Two Dimensional Gel Electrophoresis Using the O Farrell System Current Protocols in Molecular Biology 10 4 1 10 4 13 1992 Gallagher S R and Smith J A Electrophoretic separation of proteins Current Protocols in Molecular Biology F A Ausubel R Brent R E Kingston D D Moore J G Seidman J A Smith and K Struhl eds 10 2 1 10 2 21 1991 Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680 685 1970 Matsudaira P T and Burgess D R SDS microslab linear gradient polyacrylamide gel elec trophoresis Anal Biochem 87 386 396 1978 Reisfeld R A et al Acidic buffer system for resolution of cationic proteins Nature 195 281 1962 Sasse J and Gallagher S R Staining proteins in gels Current Protocols in Molecular Biology EA Ausubel R Brent R E Kingston D D Moore J G Seidman J A Smith and K Struhl eds 10 6 1 10 6 8 1991 Weber K and Osborn M The reliability of molecular weight determinators by dodecyl sulfate
19. fer miniVE complete includes 3 rectangular glass plates 1 80 6418 77 3 notched plates 2 gel modules lid tank 2 each 1 0 mm thick 10 well combs and 1 0 mm thick spacer sets Adaptors for use with Novex precast gels 4 pk 80 6421 24 Blot Module includes 3 Dacron sponges A thick 1 80 6418 96 25 sheets of blotter paper Accessories Glass plates 10 x 10 5 cm 5 pk 80 6150 87 Notched glass plates 10 x 10 5 cm 5 pk 80 6150 49 Spacers 0 75 mm thick pair 80 6149 92 Spacers 1 0 mm thick pair 80 6150 11 Spacers 1 5 mm thick pair 80 6150 30 Comb 5 well 0 75 mm thick 1 80 6140 23 Comb 5 well 1 0 mm thick 1 80 6140 42 Comb 5 well 1 5 mm thick 1 80 6140 61 Comb 9 well 1 0 mm thick microtiter 1 80 6140 80 Comb 10 well 0 75 mm thick 1 80 6138 71 Comb 10 well 1 0 mm thick 1 80 6138 90 Comb 10 well 1 5 mm thick 1 80 6139 09 Comb 15 well 0 75 mm thick 1 80 6139 47 25 26 Comb 15 well 1 0 mm thick Comb 15 well 1 5 mm thick Comb 18 well 1 0 mm thick microtiter Comb prep ref 0 75 mm thick Comb prep ref 1 0 mm thick Comb prep ref 1 5 mm thick Gel Casters Hoefer SE 200 Series SE 245 Mighty Small Dual Gel Caster 1 or 2 gels 10 x 8 10 5 cm SE 215 Mighty Small Multiple Gel Caster 5 to 10 gels 10 x 8 cm SE 275 Mighty 4 Gel Caster 2 to 4 gels 10 x 8 cm SE 235 Mighty 4 Gel Caster 2 to 4 gels 10 x 10 5 cm Electrophoresis Power Supplies EPS 300 Power Supply 300 V 400 mA 80 W Hoefer EPS 2A200 Po
20. fers should not be titrated make fresh buffer 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 56 7 increases the negative charge on proteins Diffuse band patterns Y Y Conduct the electrotransfer immediately after electrophoretic sepa ration Shorten or eliminate the equilibration step before electrotransfer or conduct equilibration in the cold room If the transfer buffer contains methanol 10 however equili brate the gel for 30 minutes to allow H to shrink fully Note Gel shrinkage may slow the migration of large molecules Y Take care that the gel does not shift once it contacts the mem brane Check that the preferred binding surface of the membrane faces the gel Inefficient binding to membrane Chemical parameters V Fix or crosslink the molecule onto the membrane according to the requirements of the nucleic acid protein or membrane type V Prepare protein transfer buffer without SDS V 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 Wear gloves when handling membranes Store membranes properly e g protect from temperature extremes and direct sunlight If proteins pass through the selected membrane try a different typ
21. igure 2 12 Preparing for elec trophoresis Note for preparative combs The side wells for standards are the same size as in the 10 well comb Note Avoid wetting the banana plugs with electrophoresis buffer A protective film of GelSealTM supplied can be applied to the plugs as a corrosion barrier 2 2 Final assembly 1 3 Make sure the sealing plate is in the half open position The arrow in Figure 2 12 indi cates the correct position er The half open N position for electrophoresis Tip To aid in loading sam ples either mark the well location with a laboratory marking pen or wet the well locating decal and apply it to the front of the glass plate so that the appropriate edge out lines the sample wells Upper buffer chamber Lower each module into the tank seating it in the locating slots The module seats prop erly in only one orientation with the banana plugs toward the center of the tank the gel facing outward Add the appropriate amount of electrophoresis buffer to the tank General guidelines add 1 2 1 6 liters of buffer to the tank when only one module is in place and 1 1 1 4 liters when two modules are in place The minimum and maximum levels are marked The minimum level ensures that the lower electrode which is approximately 2 cm from the bottom of the module is completely submerged Verify that it is The maximum level prevents buffer in the tank from
22. ions solutions and dilutions e g do not use Tris HCl instead of Tris V Ifthe required pH of a solution is exceeded do not back titrate Prepare fresh buffer V Use only stock of the highest quality Dispose of outdated acrylamide solutions V Only use freshly deionized urea Voltage or current settings V Toincrease or decrease the migration rate adjust the voltage or current by 25 50 Stained sample collects Near the buffer front V Protein or nucleic acid is not sufficiently restricted by the resolving gel increase the T Near the top of the gel when the buffer front has reached the bottom V The gel pore size is too small Decrease the 96 T of the resolving gel V Protein precipitates or the DNA becomes denatured Decrease the temperature at which the sample is prepared to 70 or less and limit exposure to heat to 1 2 minutes Smile effect on the buffer front To reduce the running temperature V Prechill the buffer V Decrease the current or voltage setting Laemmli gel guidelines 10 mA per 0 75 mm gel 15 mA per 1 5 mm thick gel V Conduct electrophoresis in the cold room V Fillthe tank to the maximum marked buffer level Bands are skewed or distorted Gel preparation V Deaerate the stacking gel solution and avoid trapping air bubbles under the comb teeth V Overlay the monomer solution with water saturated n butanol to avoid forming an uneven gel surface Sample preparation V
23. mM Tris 192 mM glycine 0 20 v v methanol pH 8 3 1 liter Tris FW 121 1 25 mM 3 09 Glycine FW 75 07 192 mM 144 g SDS FW 288 4 0 1 3 5 mM 109 Optional Adding SDS can improve transfer efficiency 1 Dissolve in 750 ml distilled water 2 Add methanol as required Depending on the membrane type selected adding methanol can improve the transfer results Because buffers containing methanol may deteriorate if stored for long periods add methanol just prior to transfer 3 Bring to 1 liter with distilled water Do not adjust the pH which should be between 8 2 and 8 4 4 Optional Chill before use 3 3 Prepare the transfer stack Transfer the sample as soon as possible after electrophoresis to minimize sample diffusion within the gel Electrophoretic transfer can be performed on as many as four mini gels at one time if two gels are placed in each of two modules 1 The transfer stack consists of the gel and membrane filter paper and three packing sponges The gel determines the size of the membrane and filter paper for each gel cut the membrane and two pieces of filter paper the same size as the gel but no larger than 8 5 x 10 5 cm Figure 3 2 Transfer stack assembly The module is color coded black cathode red anode Important Try to place the gel correctly the first time because proteins may begin to transfer imme diately once transfer has begun moving the gel will distort results or
24. onized water to 4 C before adding to the tank Place the safety lid on the tank Either orientation fits and is correct Plug the color coded leads into the jacks of an approved power supply such as the EPS 300 red to red black to black The black cathode side faces toward the center _ The red anode side faces toward the outside tank wall Note Never leave the unit unattended for more than one hour once electrotransfer has begun Note 300 350 ml Towbin buffer contains suffi cient buffering capacity for a transfer period of up to 2 hours at 300 mA 3 5 Electrotransfer Electrophoretic transfer conditions for blotting proteins in Towbin buffer 25 V for 1 2 hours 300 400 mA Important We recommend programming the power supply to hold the current setting constant to avoid possible overheating especially if no passive cooling is in place Buffer conductivity increases with increasing temperature providing a pos itive feedback that results in rapid heating If the only programming option is to hold the voltage setting constant monitor and adjust the voltage to maintain the current at or below 400 mA 3 6 After electrotransfer 1 Turn off the power supply and disconnect the leads 2 Remove the safety lid Lift out the module s and drain by inverting over a sink Avoid wetting the banana plugs with buffer 3 Open the module Remove the gels and membranes Save the packing sponges Discar
25. ophoresis Module This section describes the use of the electrophoresis module For instructions on using the blot module see page 17 2 1 Preparing the gel Both self cast and precast gels of the following dimensions fit the electrophoresis module 10 cm wide 8 to 10 5 cm long 0 75 to 1 5 mm thick For instructions for precast gels see section 2 1 2 2 1 1 Self cast gels One single gel can be cast on the module To cast several gels a multiple gel cast er such as the Hoefer SE 215 SE 235 Mighty Small 4 Gel SE 245 Mighty Small Dual Gel Caster or SE 275 Mighty Small Multiple Gel Caster is required see ordering information Clamp screws 4 1 Position the module to accept the gel sandwich Each of the three sealing elements is hinged and must be placed in the open position a Release the sealing plate by applying gentle pressure to both tabs as indicated by the arrows Then hold ing the tabs move the plate into the fully open position C b Loosen all four screws 4 5 turns in the counter clockwise direction C Swing the clamps out ward Lay the module flat on a work surface Hoefer miniVE 5 Figure 2 3 Gel sandwich assembly Figure 2 4 Placing the sandwich Assemble the gel sandwich Choose one notched plate one rectangular glass plate and two spacers Use only unchipped plates 4 to prevent leaking AF Assemble the gel sandwich as shown the notch is at the top of the sand
26. or potassium concentration The Blot Module orderea separately The Hoefer miniVE blot module ordered separately performs electrotransfers on mini format gels Each module holds up to two gels 8 2 cm wide and up to 10 4 cm long One or two modules can be run at one time 3 1 Assembly 1 Prior to use wash the tank and blot module with a dilute solution of non abrasive laboratory detergent Thoroughly rinse with water and distilled water 2 Install the gaskets Separate out two of the four strands of gaskets included with each module Open the module by unlatching both tabs Lay a gasket along the entire groove around each cup half Avoid stretching or twisting the gasket the length should just fit Gently press into place Figure 3 1 Open the cup by releasing both tabs Install a gasket into Once gaskets are in place the cup will each cup half pop open when the tabs are released A gasket fits into the groove around three sides of each cup half 17 18 3 2 Preparation 3 2 1 Optional Passive cooling Chill approximately 2 liters of deionized water to 4 C Filling the tank with chilled water serves as a heat sink during electrotransfer 3 2 2 Prepare transfer buffer Transfer buffer is required for stack assembly 250 ml and to fill each module 300 350 ml per module The recipe for the commonly used Towbin buffer is listed below The Bibliography lists sources for other buffers Towbin buffer 25
27. rreparable damage to the unit Chilled buffer provides passive cooling that can help control overheating to some degree P To clean the safety lid wipe with a damp towel or briefly rinse the underside only Do not immerse p Do not autoclave or boil this unit or any of its parts p Do not remove the screws in the clamps four full turns in the counterclockwise direction are sufficient to be able to open the clamps If this equipment is used in a manner not specified by the manufacturer the protec tion provided by the equipment may be impaired Only accessories and parts approved or supplied by Amersham Biosciences may be used for operating main taining and servicing this product A A 1 2 Informations importantes p 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 b Ne pas travailler avec un gel ou un tampon dont la temp rature d passe 75 C Une sur chauffe causerait des dommages irr parables l unit d lectrophor se En dessous de condi tions normales veuillez pr venir d une sur chauffe en refroidissant le tampon avant l utili sation p Pour nettoyer le couvercle de s curit essuyer avec un chiffon humide ou rincer uniquement le dessous Ne pas immerger p Ne pas autoclaver ou st riliser cette unit ni aucune de ses pi ces d
28. s and glass plates are not perfectly aligned against the stops use the stiff end of the Hoefer Wonder Wedge provided to press against the edges of the spacer and glass plates to position them flush against the guide Figure 2 7 foot Misalignments will cause leakage c Complete clamping by tight ening each screw firmly hand tight Do not overtighten as the plates may crack Check Important Check the bottom edges for proper alignment against the guide feet the spacer alignment The spacer must The glass plate should not protrude out of not be resting on the the sandwich head of the spacer T Tip A test for alignment involves passing a corner of the Wonder Wedge across the bot tom edge of the spacers and glass plates If an edge catch Figure 2 8 es realign Check both sides Final assembly Engage each tab in the topmost notch d Lock the sealing plate into the closed position by engaging each tab in its topmost notch Hoefer miniVE 7 Figure 2 9 Convenient module position for pouring the gel If a counterweight is required for balance either fill the tank or hang the second mod ule on the other side Tip Approximately 10 ml of monomer solution is required to cast one 1 mm thick gel 10x10 5 cm plates Hang the module from the side of the tank or stand it on the benchtop to cast the gel Tabs on the narrow side of the tank fit into slots in the module Prepare
29. s outward 4 Remove the gel from the sandwich or cassette Gently loosen and then slide away both spacers Slip an extra spacer or the Hoefer Wonder Wedge into the bottom edge to prevent breaking the ears of the notched plates and separate the plates If using precast gels follow gel manufacturer s instructions Carefully lift the gel from the plate and lay it into a tray containing stain fixative or transfer buffer 5 Clean the unit as described in Section 2 5 13 14 2 5 Care and Maintenance D Do not autoclave or heat any part above 75 C D Do not immerse the safety lid in any liquid D Do not use organic solvents strong or oxidizing cleaning solutions abra sives or strong acids or bases on any part of the instrument Immediately after each use rinse the tank and modules with water and then rinse thoroughly with distilled water Handle the module with care to prevent damage to the banana plugs Allow to air dry Wipe the lid with a damp cloth If necessary briefly rinse the underside of the lid with water Clean glass plates and spacers with a dilute solution of a laboratory detergent such as RBS 35 then rinse thoroughly with tap and distilled water Glass plates can be treated with but not stored in acid cleaning solutions A final wipe with isopropanol will remove any GelSeal residue 2 6 Troubleshooting Unusually slow or fast run Reagent and solution factors v Check recipes gel concentrat
30. tegory Pollution degree Dimensions w x d x h Weight tank lid and two gel modules Product certifications 10 0 cm wide x 8 to 10 5 cm long 1 7 liters with one module in place 1 2 liters with two modules in place 600 V 25 W per electrophoresis module 350 ml per module 1 7 liters with one module in place 1 2 liters with two modules in place 15 W per blot module 400 mA 75 C For use only with dilute aqueous solutions between pH 2 and pH 12 Not compatible with organic solvents or concentrated alco hols acids bases and oxidizing agents Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Il 2 19 2 x 17 2 x 18 8 cm 7 6 x 6 8 x 7 4 in 1 2 kg 2 65 bei EN61010 1 UL3101 1 CSA C22 2 1010 1 CE This declaration of conformity and the warranty are only valid when the instrument is p used in laboratory locations p used within the conditions specified in the User Manual 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 by Amersham Biosciences 1 2 Important Information b 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 Do not operate with the gel or buffer temperature above 75 C Overheating will cause i
31. the monomer solution and pour the resolving gel Caution Acrylamide is a neuro toxin Always wear gloves and observe all laboratory safety pro cedures Pipet the solution into the sand wich slowly so that it flows along a spacer taking care not to trap any air pockets No stacking gel fill the solution to the desired level and then insert a comb at a slight angle into the sandwich taking care not to trap air under the teeth For a 1 cm stacking gel below the wells fill to 3 cm below the top of the rectangular glass plate Overlay each gel with a thin layer of water saturated n butanol water or diluted gel buffer to prevent exposing the monomer solution to oxygen Apply the overlay solution 100 pl slowly to one side of the sandwich near the spacer using a glass syringe fitted with a 22 gauge needle Allow the solution to flow across the surface unaided Allow a minimum of one hour for the gel to polymerize If a comb is in place remove it by carefully pulling on the comb while gently rocking it back and forth to break the vacuum Rinse the wells with elec trophoresis buffer to remove any unpolymerized acrylamide If an overlay was applied rinse the sandwich several times with double dis tilled water to remove it Invert the module to drain For instructions on pouring a stacking gel see section 2 1 3 If the gel is ready for electrophoresis move the sealing plate into the half open position Apply gen
32. tle pressure to both tabs and lock them into the lower notch 2 1 2 Precast gels 1 See Section 2 1 1 step 1 to prepare the electrophoresis module Figure 2 10 Novex gel cassettes only Adaptor Installing adaptor sleeves ordered separately must be sleeves installed on each clamp Slide an adap tor sleeve on each pressure plate from the bottom as shown in Figure 2 10 The sleeve fits in only one orientation and snaps into place when fitted prop erly Slide the adaptor sleeve 2 Follow the manufacturer s instructions over the pressure plate to prepare the gel for electrophoresis This may involve removing tape or breaking off the sealing edge from the bottom of the cassette 3 Remove the comb and rinse the wells with electrophoresis buffer to remove any unpolymerized acrylamide Tip To aid in sample loading mark the well locations with a laboratory marking pen 5 Figure 2 11 Securing the cassette ils g 4 Position the cassette on the module Orient the cassette so that the notched T F side is against the gasket and the wells ei are at the top of the module Center the cassette within the guides at both sides of the module a 5 Secure the cassette Swing each clamp into position over the sides of the cas sette Tighten each screw alternating to apply even pressure until the cassette is secure The gasket around the upper buffer chamber should be fully com pressed to provide a seal but the
33. 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 attir er l attention de l utilisateur sur des instructions importantes de fonction nement ou de maintenance Le symbole de l clair dans un triangle quilat ral a pour objet d attirer l atten tion de l utilisateur sur un danger d expo sition la haute tension Tous vos commentaires sur ce manuel seront les bien venus et veuillez les adresser Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA Amersham Biosciences se r serve le droit d ef fectuer des modifications de ces sp cifications sans aucun pr avis Wichtige Benutzerinformationen F r ein vollst ndiges Verst ndnis und eine sichere Handhabung dieses Produktes ist es notwendig daf der Benutzer dieses Handbuch vollst ndig durchliest Ein Ausrufezeichen in einem gleichseiti gen 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 hinweisen Wenn Sie Anmerkungen zu diesem Handbuch haben dann senden Sie diese bitte an Amersham Biosciences Marketing Department 654 Minnesota Street San Francisco CA 94107 USA
34. wer Supply 200 V 2000 mA 200 W Gel Drying System Hoefer EasyBreeze Air Gel Dryer 115 V Hoefer EasyBreeze Air Gel Dryer 230 V Protein Molecular Weight Markers LMW Marker Kit HMW SDS Marker Kit HMW Marker Kit PlusOne Chemicals Acrylamide PAGE 250 g Acrylamide PAGE 1 kg Acrylamide PAGE 40 solution 1000 ml Acrylamide IEF 250 y Acrylamide IEF 4096 solution 1000 ml N N methylenebisacrylamide 100 g N N methylenebisacrylamide 296 solution 1000 ml Ammonium persulfate 25g TEMED 25 ml Tris 500 g Boric Acid 500 g EDTA disodium salt 1009 Urea 500 y Silver Staining Kit Protein Silver Staining Kit DNA 4 4 80 6139 66 80 6139 85 80 6140 04 80 6141 56 80 6141 75 80 6141 94 80 6146 50 80 6142 51 80 6151 06 80 6146 12 18 1123 97 80 6406 99 80 6121 61 80 6121 80 17 0446 01 17 0615 01 17 0445 01 17 1302 01 17 1302 02 17 1303 01 17 1300 01 17 1301 01 17 1304 02 17 1305 01 17 1311 01 17 1312 01 17 1321 01 17 1322 01 17 1324 01 17 1319 01 17 1150 01 17 6000 30 Amersham Life Science Products Rainbow colored molecular weight markers low molecular weight Rainbow colored molecular weight markers high molecular weight Rainbow colored molecular weight markers full range 1 1 1 ECL Protein molecular weight markers with Strep HRP 1 ECL Western blotting molecular weight markers 1 ECL Plus Western blotting detection system 1 ECL Western blotting reagents sufficient for 1000 cm of membrane 1
35. wich and the spacer ridges align along the glass plate edges on the sides of the sandwich IMPORTANT PROPER ALIGNMENT IS ESSENTIAL IN STEPS 3 AND 4 TO PREVENT LEAKAGE TAKE CAREFUL NOTE OF THE ALIGNMENT TIPS Place the sandwich on the module Take care to square the three sealing sides of the sandwich This can be done by holding the sandwich like a deck of cards and gently tapping the bottom against a flat surface Tip Once the sandwich is carefully aligned handle it with the flat sides firmly between your thumb and fingers near the notch Notched plate side down lay the sandwich on the module fitting it within the guides at both sides a and against the guide feet at the bottom b The notched plate is against the module with the notch at the top The gel sandwich fits within guides at the sides and aligns flush against the guide feet at the bottom of the module Section 2 The Electrophoresis Module 4 Seal the sandwich Figure 2 5 a While gently holding the Positioning the clamps sandwich against the module swing one clamp into position over the spacer taking care not to bump the sandwich out of alignment Turn each screw alternating to keep the pres sure even until the clamps are loosely secured and will allow the spacers to be adjusted if necessary Repeat on the other side Figure 2 6 Checking the alignment b Important Check the alignment at the bottom of the sandwich If the spacer
36. y red to red black to black The minimum power supply rating is 250 V 50 mA constant current or constant voltage Recommended power supply Ka EPS 300 12 Important After initial monitoring do not leave the unit unattended for more than 30 minutes before checking the buffer level and the progress of the bands Important Always disconnect the high voltage leads from the power supply before removing the lid from the unit 2 3 Electrophoresis For optimal resolution electrophoresis should be started immediately after sample loading Gels may be run at either constant current or constant voltage For Laemmli SDS separations the recommended voltage range is 100 250 V and should not exceed 300 V If running gels at constant current the current should be 10 20 mA per gel depending on gel thickness 10 mA for 0 75 mm 15 mA for 1 5 mm Check progress after 5 minutes and again after half an hour monitoring the posi tion of the tracking dye The run is complete when the tracking dye reaches the bottom of the gel 2 4 After electrophoresis 1 Turn off the power supply and disconnect the leads 2 Remove the safety lid and lift out the module s 3 Release each gel sandwich or cassette from the module Move the sealing plate to the fully open position by pressing inward on both tabs and guiding the plate to open out Then unscrew all four screws 4 5 turns in the coun terclockwise direction Swing the clamp

miniVE - GE Healthcare Life Sciences

Contents

Download Pdf Manuals

Related Search

Related Contents