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Multiphor ll Electrophoresis System

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1. Multiphor Il Electrophoresis Unit application kits accessories and replacement parts MultiTemp III EPS and GPS Power Supplies Precast gels and buffer strips Molecular weight and pl markers Ampholine and Pharmalyte carrier ampholytes Gel casting and electrophoresis chemicals Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Safety information 2 2 Safety information To avoid any risk of injury the instrument should be operated only by properly trained personnel and always in accordance with the instruction provided Read this entire manual before using the instrument WARNING The instrument is designed for indoor use onlu WARNING Do not operate tthe system in extreme humidity above 95 RH Avoid condensing by equilibrating to ambient temperature when taking the unit from a cooler to a waremer environment WARNING Always check the wires for damage before using the unit WARNING Always check that the electrodes are properly connected before using the lid WARNING Always connect the lid according to the mounting instruction WARNING Always connect the cables to the Power supply BEFORE turning the Power Supply ON WARNING Always TURN OFF the Power Supply before removing the lid WARNING Do NOT use concentrated acids bases or halogenated and aromatic hydrocarbons WARNING Only use water or coolant
2. Coomassie staining On completion of the electrophoresis immediately immerse the gel in Staining Tray 1 using the solutions and times indicated in table below The staining and destaining steps should be carried out on a shaking table See section 4 9 for stock solutions Each step requires 250 ml of solution Step No Solution Time min Temp C 1 Fixing solution C 20 23 2 Destaining solution 2 23 3 Staining solution K 10 60 4 Destaining solution 20 23 5 Destaining solution 30 25 6 Preserving solution L 10 23 The staining solution should be heated to 60 C and poured over the gel No further heating is necessary Destain the gel using several changes of destaining solution 1 until the background is clear Change the solution frequently particularly at the beginning in order to speed up the destaining To preserve the gel soak a cellophane sheet in preserving solution L Place it on the gel surface Remove any air bubbles and wrap the excess cellophane around the glass plate An additional glass plate may be placed underneath during drying to stop the cellophane from shifting or wrinkling Leave the gel at room temperature until it is completely dru Silver staining Silver staining is performed essentially as described by J Heukeshoven and R Dernick Electrophoreses Forum 1986 22 27 On completion of the electrophoresis immediately immerse the gel in Staining Tray 1 using the solutions and times indicated in t
3. 5 Operation h 3 Carefully place the filter papers onto the anode electrode When forming the first transfer sandwich soak a further layer of three filter papers in anode solution S see Section 4 9 Stock solutions using the same method as above Place them on top of the six filter papers on the anode electrode plate again taking care to avoid trapping air bubbles When using a continuous buffer system all filter papers cathodic and anodic are wetted in the same solution Note To obtain optimal transfer of molecules from the gel care should be taken to avoid trapping air bubbles at all stages of the assembly of the transfer sandwiches 4 Cut the gel loose from the support film using FilmRemover Do not move the gel leave it on FilmRemover Wet the immobilizing membrane in electrode solution S and carefully place it on top of the gel on the FilmRemover Note Wear gloves to avoid contamination of the membrane 38 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 5 Loosen the support film from FilmRemover by pressing the handle and carefully lift the whole sandwich with the support film immobilizing membrane and gel Turn it over support film up immobilizing membrane down and place it on the layer of three filter papers on the anode Carefully remove the support film If air bubbles become trapped under the gel wet the surface of the gel with a few drops of electrode solution a
4. 34 5 5 2 D electrophoresis using Immobiline DryStrip and ExcelGel SDS This chapter gives a brief description of the 2 D two dimensional electrophoresis method using Immobiline DryStrip and ExcelGel For more detailed information on running conditions please refer to the instructions supplied with Immobiline DryStrip Kit Isoelectric focusing using IMmobiline DryStrip makes true isoelectric focusing possible and significantly improves the reproducibility of the spot distribution along the pH gradient axis of 2 D maps Immobiline DryStrip also makes it possible to obtain distinct protein spots even of basic proteins The Immobiline DryStrip Kit facilitates sample application running and equilibration of Immobiline DryStrip for the first dimension of 2 D electrophoresis The kit includes the accessories necessary to run up to 12 Immobiline DryStrip strips simultaneously on Multiphor Il Sample cup loading allows the application of up to 100 ul on each Immobiline DryStrip See application Note 80 1443 47 for unultiple miniformat 2 D lectrophoresis using ExcelGel 2 D 12 5 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 5 6 Electrophoretic transfer Introduction The method of horizontal semi dry electrophoretic transfer gives fast even and efficient transfer of proteins from a gel to an immobilizing membrane The resulting membranes may be used for a wide range of applications including general pr
5. Check the electrode polarity Check the pH of the applied electrode strips Use insulating fluid under the gel and check for air bubbles Cut the electrode strips shorter than the edge of the gel If necessary blot the pooled liquid Check pH of the strips and polarity of the plugs in the power supply Reverse polarity if the strips have been incorrectly applied should be acid at anode Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Symptom Skewed or wavy bands Cause Localized gradient disturbances due to excessive salt Unevenly wetted electrode strips Electrode strips too short Trouble shooting guide to electrophoretic transfer Symptom Incomplete transfer Poor transfer Inefficient transfer Cause Gel concentration too high Methanol present in transfer buffer Transfer time too short Field strength too low Too low charge mass ratio Air trapped between gel and membrane Too low binding efficiency molecules migrate from the gel but pattern is faint Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Trouble shooting 8 Remedy Reduce the salt content of the sample by gel filtration using PD 10 columns pre packed with Sephadex G 25 Salt content should be lt 50 mmol l Too much ammonium persulphate may also cause wavy bands Electrode strips must be evenly wetted and be neither too wet nor too dry The strips should be cut just short of the
6. 15 20 pl volumes of sample solution on each piece To apply larger volumes use 2 or 3 pieces stacked or placed end to end for each sample applied If smaller volumes are used trim the paper proportionally before applying it to the gel Remove the pieces after completing approximately half the total focusing time 3 Very small sample volumes e g 2 ul can be applied as droplets directly onto the dry gel surface To determine the pH gradient prepare and apply pl markers according to the instruction sheet included in the pl Marker Kit Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 29 5 Operation 30 Starting the IEF run Place the electrode holder in the shallow depressions on the Multiphor Il unit Align the electrodes with the centre of the electrode strips by loosening the clamping nuts and sliding the electrodes to the appropriate position Retighten the clamping nuts Lift the electrode holder slightly and reposition the supporting feet over thedeep holes Lower carefully so that the electrodes rest on the electrode strips As the anodic pin and socket connectors are different for electrophoresis using buffer chambers and isoelectric focusing a red bridging cable has been provided During isoelectric focusing the socket on the bridging cable MUST be attached to the pin connector Connect the two electrodes to the buffer tank using the spring loaded cables on the electrodes Connect th
7. Remove any excess solution with a paper tissue Soak the electrode strips evenly in the appropriate electrode solution see table above approximately 3 ml strip The surface of the strips should look wet Remove excess solution with a tissue paper Beas A Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Apply the electrode strips to the long edges of the gel Make sure the electrode strips are applied with the correct polarity Use sharp scissors to cut off the strips which protrude beyond the ends of the gel Sample application Some proteins may be pH sensitive they may precipitate at specific pH values and better separation may be obtained when the sample is applied at a specific position in the pH gradient This can be tested by appluing the sample at various positions across the pH gradient There are three different methods for sample application The method of choice depends primarily by the sample and the volume to be applied 1 IEF SDS applicator strip for 5 20 ul sample volumes This applicator strip makes sample loading quick and simple especially when a large number of samples are to be applied Check that the contact between the gel and the applicator strip is uniform Leave the applicator strip on the gel during focusing 2 Sample application pieces Place the dry pieces on the Ampholine PAGplate surface at the desired positions s in the gradient Using a micropipette apply
8. and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment 156 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 47 6 Maintenance 48 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 7 Technical specifications Maximum Voltage Maximum Power Max pressure cooling plate Dimensions Environment Material of wetted parts Chemical resistance Compliance with standards Safety standards EMC standards Technical specifications 7 3500 Vp p 1750 V with reference to Ground 100 W 0 5 bar 16x31x40 cm 4 40 C 20 95 relative humidity The wetted parts are resistant to solvents commonly used in electrophoresis and solutions containing inorganic and organic acids alkalis and alcohols The declaration of conformity is valid for the instrument only if it is e used in laboratory locations e used in the same state as it was delivered from GE Healthcare except for alterations described in the User Manual e connected to other CE labelled GE Healthcare modules or other products as recommended This product meets the requirement of the Low Voltage Directive LVD 73 23 EEC through the following harmonized standards e EN 61010 1 e IEC 61010 1 e CAN CSA C22 2 No 61010 1 e UL61010 1 This device meets the requirements of the EMC Directive 89 336 EEC through the
9. distribute it evenly with a tissue Leave it to dry for a few minutes Rinse the glass plate with distilled water and remove water drops by shaking or wiping lightly with a tissue Leave the glass plate to dry When using the 1 mm thick glass plate as the gel support simply lay it directly on top of the 3 mm thick glass plate If the gel is to be permanently bound to the 1 mm thick glass plate coat the plate with Bind Silane before preparing the mould Note For this operation use gloves and a fume hood Pour about 2 ml of diluted Bind Silane onto the glass plate and distribute it evenly with a tissue Leave the glass plate to dry for a few minutes rinse with distilled water and leave to dru 12 When using GelBond PAGfilm pour a few ml of water on to the 3 mm thick glass plate and lay the film over it with the hydrophilic side up see Instructions supplied with the film Centre the film on the glass plate Beginning at one end use the roller to apply even pressure over the film surface in order to eliminate air bubbles and seal the film to the plate with a minimum of water Remove any excess water with a tissue Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 57 9 Multiphor Il application kits and accessories 58 Form the mould by placing the U framed glass plate in position and clamp together using four FlexiClamps Note Gloves must be worn to protect the user from contact with the toxic acrylami
10. edges of the gel Remedy Use reversible cross linkers e g DATD BAC DHEBA and depolymerize gel before transfer Lower monomer concentration Convert molecule to smaller form by limited digestion with proteases for proteins or with nucleases or acid hydrolysis for nucleic acids Remove methanol from transfer buffer Increase transfer time Increase field strength Change transfer buffer pH further away from molecules pl Add 0 1 SDS Carefully push out all air bubbles from the layers of the transfer sandwich Use different immobilizing membrane DEAE NC DBM DPT Immobilizing membrane needs to be activated Remove interfering substances denaturants detergents Raise lower salt concentration Raise lower pH 53 8 Trouble shooting Symptom 54 Cause Field strength too high e g low Mol Wt DNA Transfer time too long Pore size too large Remedy Lower field strength Shorten transfer time With nitrocellulose use smaller pore filters 0 1 or 0 7 um Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Multiphor Il application kits and accessories 9 9 Multiphor Il application kits and accessories This section describes the contents of the Multiphor Il application kits and accessories and provides instructions for assembly and use For experimental details including preparation of samples and stock solutions running conditions staining and preserving procedures see Chap
11. following harmonized standards e EN 61326 emission and immunity e EN 55011 GR 2 Class A emission e This device complies with part 15 of the FCC rules emission Operation is subject to the following two conditions 1 This device may not cause harmful interference 2 This device must accept any interference recevide including interference that may cause undesired operation Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 49 7 Technical specifications 50 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK 8 Trouble shooting Trouble shooting 8 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 equipment l WARNING Turn OFF the power supply before opening the lid Trouble shooting guide to PAGE Symptom Cause No current reading Safety plug improperly inserted in power supply outlet Pin and socket connection from electrode to base incomplete Anode bridging contact disconnected Banana plug connection in safety lid not completed Electrode holder not seated properly Poor contact between the electrodes and electrode strips Uneven migration of Bad electrical contact between the gel the dye front and the wicks and electrodes Poor cool
12. in the grooves in the wall closest to the centre of the unit The wicks lie at the outer edge of the buffer chamber Place the cathode electrophoresis electrode in the left buffer chamber and the anode in the right buffer chamber Fill each chamber to the moulded line indicating 1 liter volume with buffer solution When running 120 x 250 mm gels pour 1 2 liters of buffer into each chamber to ensure adequate buffer contact with the wicks Replace the cooling plate making sure that the electrode socket connectors lie to the front and that the connecting cable is clear of the feet on the plate Disconnect the anode bridging connector for isoelectric focusing and connect the electrodes to their respective pins When performing immunoelectrophoresis or agarose electrophoresis center a small 84 x 94 mm glass plate on the dry cooling plate and attach a strip of tape along the width of the cooling plate in alignment with the edges of the glass plate These stop the small gels from shifting during application of the wicks and ensure that they are centred between the two buffer chambers during electrophoresis Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 25 5 Operation 26 Switch on MultiTemp Ill thermostatic circulator and set the desired temperature normally 10 C for PAGE or agarose electrophoresis and 15 C for SDS PAGE 15 minutes before starting the experiment To ensure efficient heat transfer from the gel d
13. plate may be placed underneath during drying to stop the cellophane from shifting or wrinkling Leave the gel at room temperature until it is completely dry 5 4 Isoelectric focusing using CleanGel IEF CleanGel IEF is a washed and dried polyacrylamide gel optimized for analytical isoelectric focusing Prior to use the dried gel is rehydrated in a solution containing Ampholine or and Pharmalyte Additives such as urea and or detergents can also be added at this stage CleanGel IEF is rehydrated in a flat tray GelPool to a thickness of 0 43 mm Instructions are supplied with the product Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 ii u 4 Sample application Use the same methods as for Ampholine PAGplate see Section 4 4 Running conditions In principle CleanGel is run in the same way as Ampholine PAGplate except no electrode strips are used The electrodes rest directly on the gel surface Recommended running conditions for one CleanGel IEF 3 10 Phase Voltage Current Power Time V MA W min Prefocusing 700 12 8 20 Sample entrance 500 8 8 20 Isoelectric focusing 2000 14 14 90 Band sharpening 2500 14 18 10 Note If only half a gel is used halve the current and power settings Detection Methods Use the detection methods described for Ampholine PAGplate see Section 4 4 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 33 5 Operation
14. two transfer sandwiches side by side The operating procedures for NovaBlot Kit and FilmRemover are described and illustrated in Sections 4 8 and 7 13 respectively Kit contents Code No 18 1016 86 Designation Code No NovaBlot Electrode cathode 18 1019 86 NovaBlot Electrode anode 80 1257 87 Electrode Paper NovaBlot 200 x 250 mm 500 pkg 80 1106 19 Cellophane Sheets 210 x 320 mm 50 pkg 80 1129 38 Optional accessories Designation Code No FilmRemover 18 1013 75 Nitrocellulose 0 20 um 150 x 200 mm 15 pkg 80 1098 91 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 61 9 Multiphor Il application kits and accessories 62 9 6 FilmRemover FilmRemover is used for removing backing from a gel before electrophoretic transfer Polyacrulamide or agarose gels with a thickness between 0 1 mm and 5 0 mm anda maximum gel size of 200 x 245 mm can be used E Detailed instructions for the use of FilmRemover are available in the instruction manual provided with the product Unit contents 18 1013 75 Designation FilmRemover basic unit Lever and Wire Assembly 3 pkg Instruction Manual Nitrocellulose 0 20 um 150 x 200 mm 15 pkg Nitrocellulose 0 45 um 150 x 200 mm 15 pkg ProBind 45 NC 0 45 um roll 0 2 x 3 0 m GeneBind 45 nylon 0 45um roll 0 2 x 3 0 m Code No 80 1316 21 18 1013 79 80 1316 37 80 1098 91 80 1098 90 80 1247 86 80 1247 87 Multiphor Il Electrophoresis Sy
15. 0 pkg Safety Lid Electrode Holder EPH IEF Electrode cathode EPH IEF Electrode anode User Manual and Application Package Code No 18 1122 25 18 1026 40 18 1103 46 80 1106 58 80 1106 56 18 1104 26 18 1104 27 18 1122 26 80 1106 55 80 1121 52 80 1121 53 18 1103 44 The buffer tank is made of polypropylene which is resistant to nearly all chemicals at room temperature The buffer tank contains four pin contacts Viewing the buffer tank from the front the cathode pins are located to the left and the anode pins to the right The larger pins are for connection to the safety lid and complete the electrical circuit when the lid is in position Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 11 3 Description of parts 12 The small left hand pin is used to connect the EPH IEF or card mounted cathode electrodes The small pin on the right connects to the card mounted anode or the EPH IEF cathode via the red lead mounted on the unit The buffer tank holds the four adjustable levelling feet supports the cooling plate and is covered during electrophoresis with the safety lid The buffer tank includes four buffer chambers each with a 1 liter capacity allowing the user to choose one of two orientations for electrophoretic runs The safety lid contains electrode leads apertures for voltage measurement and a safety interlock The well recessed cathode connector black and anode connector r
16. 00 Total 16 45500 The optimal total number of Volt hours for these pH gradients depends on the type of sample sample load ug and sample volume Option 2 Using a Manual Power Supply The power supply should run at constant voltage with the parameters set as below All steps are run at 10 C Running conditions for Immobiline DryStrip pH 3 10 110 mm Phase Voltage Current Power Time Vh V MA W h 1 300 1 5 1 300 2 1400 1 5 14 15 20000 Running conditions for Immobiline DryStrip pH 3 10 L 180 mm Phase Voltage Current Power Time Vh V MA W h 1 500 1 5 1 500 2 3500 1 5 15 16 55000 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 45 5 Operation 46 Running conditions for Immobiline DryStrip pH 3 10 NL 180 mm Phase Voltage Current Power Time Vh V MA W h I 500 1 5 1 500 3500 1 5 13 45000 Running conditions for IMmobiline DryStrip pH 4 7 110 mm Phase Voltage Current Power Time Vh V MA W h 1 300 1 5 1 500 2 2200 1 5 135 29700 Running conditions for Immobiline DryStrip pH 4 7 180 mm Phase Voltage Current Power Time Vh V mA W h 1 500 1 5 1 500 2 3000 1 5 14 5 43500 Second dimension Running conditions for ExcelGel XL SDS gradient 12 14 Step Voltage Current Power Time Temp V mA W min C 1 1000 20 40 45 15 2 1000 40 40 DS o 3 1000 40 40 160 15 Running conditions for ExcelGel SDS gradient 8 18 Step
17. 002 74 Gradient Maker SG 100 100 ml 80 6196 09 Glass Plate 125 x 260 mm 1 0 mm U frame 2 pkg 80 1106 91 Glass Plate 125 x 260 mm 2 0 mm U frame 2 pkg 80 1106 92 Tape Dymo 0 25 x 9 mm 3m 80 1129 50 Template 125 x 260 mm 10 pkg 80 1129 55 The 3 mm thick glass plate is used as a support either for the 1 mm glass plate or GelBond PAGfilm The mould comprising the 3 mm glass plate 1 mm glass plate or GelBond PAGfilm and glass plate with U frame is clamped together using four FlexiClamps To prepare a slot former for individual sample slots a glass plate with Uframe tape 0 25 x 9 mm and a template should be used One or several layers of tape can be applied to the glass plate For instance 3 layers of 5 x 3 mm will make a sample slot for 10 20 ul of sample Wash the glass plate with detergent rinse with distilled water and dry with a paper tissue Using the template as a guide apply the tape 30 mm from the open edge of the U framed glass plate avoiding air bubbles Check that all edges of the tape are cut perfectly even Leave the slot former over night to ensure that the tape adheres completely 56 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Multiphor Il application kits and accessories 9 To prevent the gel from sticking to the U framed glass plate coat the plate with Repel Silane Note For this operation use gloves and a fume hood Pour about 2 ml of Repel Silane onto the glass plate and
18. 2 16 949 2 mg 1 80 1129 83 MW range 14 000 94 000 200 ug vial 10 17 0446 01 MW range 53 000 212 000 200 ug vial 10 17 0615 01 MW range 67 000 670 000 200 ug vial 10 17 0445 01 Broad pl kit pH 3 5 9 3 17 0471 01 Low pl kit pH 2 8 6 5 17 0472 01 High pl kit pH 5 2 10 3 17 0473 01 Carbamulute calibration kit 17 0582 01 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK TI 10 Ordering information Te 10 6 Carrier ampholytes Product Quantity Code No Ampholine Ampholine preblended pH 3 5 9 5 25 ml 80 1127 15 Ampholine preblended pH 4 0 6 5 25 ml 80 1127 17 Ampholine preblended pH 5 0 8 0 25 ml 80 1127 19 Ampholine pH 3 5 10 0 25 ml 80 1125 87 Ampholine pH 3 5 5 0 25 mi 80 1125 89 Ampholine pH 4 0 6 0 25 mi 80 1125 90 Ampholine pH 5 0 7 0 25 ml 80 1125 91 Ampholine pH 5 0 8 0 25 ml 80 1125 92 Ampholine pH 6 0 8 0 25 ml 80 1125 93 Ampholine pH 7 0 9 0 25 ml 80 1125 94 Pharmalyte Pharmalyte pH 3 10 25 ml 17 0456 01 Pharmalyte pH 2 5 5 25ml 17 0451 01 Pharmalyte pH 4 6 5 25 ml 17 0452 01 Pharmalyte pH 5 8 25 ml 17 0453 01 Pharmalyte pH 8 10 5 25 ml 17 0455 01 Pharmalyte pH 4 2 4 9 25 ml 17 0562 01 Pharmalyte pH 4 5 5 4 25 ml 17 0563 01 Pharmalyte pH 5 6 25 ml 17 0564 01 Pharmalyte pH 6 7 7 7 25 ml 17 0566 01 Immobiline Immobiline Il pK 3 6 10 ml 80 1255 70 Immobiline Il pK 4 6 10 ml 80 1255 71 Immobiline Il pK 6 2 10 ml 80 1255 72 Immobiline Il pK 7 0 10 ml 80 1255 73 Immobiline Il pK 8 5 10 ml 80 12
19. 5 20 ul 18 1002 26 This applicator strip is recommended for use with PAGIEF gels and SDS gradient gels without preformed slots e g ExcelGel SDS gradient 8 18 Up to 52 samples with a sample volume of 5 20 ul can be applied in each well The applicator strip is Made of flexible silicone and is applied directly on the gel surface 64 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Multiphor Il application kits and accessories 9 Designation Code No EPH IEF Sample Application Foil 24 samples 2 4 pl 80 1129 47 This application foil with narrow slits is recommended for electrophoresis and IEF in agarose gels Up to 24 samples can be applied with a sample volume of 2 4 ul in each slit The foil is applied directly on the gel surface Designation Code No EPH Electrode anode long 80 1122 20 EPH Electrode cathode long 80 1122 19 The electrophoresis electrodes are designed for use with the buffer vessels at the side of the buffer tank allowing electrophoresis along the width of the cooling plate Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 65 9 Multiphor ll application kits and accessories 66 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK 10 Ordering information Ordering information 10 WARNING Only spare parts approved or supplied by GE Healthcare may be used for maintaining and servicing of MULTIPHOR ll 10 1 Multiphor Il
20. 55 74 Immobiline Il pK 9 3 10 ml 80 1255 75 Each bottle contains a ready to use 0 200 0 004 M solution Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK 10 7 PlusOne electrophoresis chemicals PlusOne electrophoresis chemicals Ordering information 10 Product Use Quantity Storage Code No Gel casting chemicals Acrylamide IEF IEF PAGE Sequencing 250 9 A 17 1300 01 Acrulamide IEF 40 solution IEF PAGE 1000 ml D 17 1301 01 ReadyMix IEF IEF 41 5 g1 C 17 1309 01 ReadySol IEF T40 C3 IEF 1000 ml D 17 1310 01 Acrylamide PAGE PAGE 250g A 17 1302 01 Acrylamide PAGE PAGE 1000 g A 17 1302 02 Acrylamide PAGE 40 Solution PAGE 1000 ml D 17 1303 01 ReadySol DNA PAGE T40 C5 PAGE Sequencing 1000 ml D 17 1308 01 N N Methulene bis acrylamide IEF PAGE Sequencing 230 C 17 1304 01 N N Methylene bis acrylamide IEF PAGE Sequencing 100 g C 17 1304 02 N N Methylene bis acrylamide 2 solution IEF PAGE Sequencing 1000 ml D 17 1306 01 Ammonium persulphate IEF PAGE Sequencing 259 E 17 1311 01 TEMED IEF PAGE 25ml G 17 1312 01 Sequencing Buffers Tris PAGE Sequencing 500 g A 17 1321 01 Boric acid PAGE Sequencing 500 g A 17 1322 01 EDTA di sodium salt PAGE Sequencing 100 g A 17 1324 01 Glycine PAGE Sequencing 500 g A 17 1323 01 Additives and sample treatment Urea IEF PAGE Sequencing 500 9 B 17 1319 01 Formamide IEF PAGE Sequencing 250 ml B 17 1320 01 Dithiothreitol IEF SDS PAGE 1 0 9 E 17 1318 01 Dithiothreit
21. A cm A transfer time of approximately 1 hour is normal Note The current is calculated using the surface area total length x width of the transfer sandwiches and this calculation applies irrespective of the number of transfer sandwiches in the stack Note For transfer times longer than one hour turn off the power supply remove the safety lid and carefully lift the cathode top electrode without disturbing the filter papers or gel Carefully pour on additional transfer buffer to re wetting the filter paper Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 10 When the transfer is complete turn off the power supply and disconnect NovaBlot from the power supply Remove the safety lid and the upper cathode electrode Carefully disassemble the transfer sandwiches and remove the immobilizing membranes for analysis If necessary save and stain the gel to monitor the transfer efficiency Clean the electrodes with distilled water Note Always turn off the power supply before opening the safety lid Although user safety is not endangered arcing may damage the contacts Detection methods Following electrophoretic transfer the membrane can be stored stained or probed immediately Further reading Electrophoresis in Practice A guide to theory and practice Westermeier R Ed 1993 VCH Verlagsgesellschaft mbH Weinheim Westermeier R Beisiegel U Electrophoresis 7 1 18 1986 Kyhse
22. AK Operation 5 Connect the socket of the cathode electrode to the pin at the front of the unit and the anode pin to one of the sockets at the back Place the safety lid in position by matching the extensions on the back of the lid with the openings on the base unit Using the extensions at the back as a hinge connect the male and female banana plugs by pressing down firmly on the front of the lid Connect Multiphor Il to the power supply Follow the recommended electrical settings and running times given in the instructions supplied with the precast gel Running conditions Voltage Current Power Time V mA W min Run 600 50 30 13 Approximate time or until the Bromophenol Blue front reaches the anode buffer strip When the Bromophenol Blue front has reached the anodic buffer strip electrophoresis is complete and should be stopped Ending the run Turn off the power supply Disconnect the Multiphor Il unit from the power supply Remove the safety lid from the unit Carefully remove the electrode holder Gently pull the strips from the gel and continue with detection techniques as required IN WARNING Always TURN OFF the power supply before opening the safety lid Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 23 5 Operation 24 Detection For automated silver and Coomassie staining of polyacrulamaida gels see Protocol guide Hoefer Automated Gel Stainer 80 6343 34
23. Andersen J J Biochem Biophys Meth 10 203 1984 Naaby Hansen S Linme A O F Bog Hansen T C Bjerrum O J in Lectins Biology Biochemistry Clinical Biochemistry Walter de Gruyter amp Co Berlin amp New York 241 1985 Handbook of immunoblotting of proteins ed Bjerrum O J amp Heegaard N H H CRC Press Florida USA Volume 1 Technical descriptions Volume Il experimental and clinical applications Hancock K and Tsang V S W Anal Biochem 133 157 162 1983 Towbin H Staehlin T Gordon J Proc Natl Acad Sci USA 76 4350 4354 1979 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 41 5 Operation 42 5 7 Stock solutions B Sample buffer 0 050 mol l Tris HAc pH 7 5 Dissolve 0 3 g Tris in 40 ml distilled water Carefully adjust to pH 7 5 with HAc approximately 0 14 ml Make up to 50 ml with distilled water Add 0 4 g SDS and a few grains of Bromophenol Blue Immediately before use add 40 mg of DTT C Fixing solution Ethanol Acetic acid HAc Make up to 1000 ml with distilled water D Incubation solution Ethanol Sodium acetate Glutaraldehude 25 w v Sodium thiosulphate Na S 0 x 5H 0 Make up to 250 ml with distilled water E Silver solution Silver nitrate Formaldehyde Make up to 250 ml with distilled water F Developing solution Sodium carbonate Formaldehyde Make up to 250 ml with distilled water G Stop solution EDTA Na
24. GE Healthcare Multiphor ll Electrophoresis System User Manual Important user information All users must read this entire manual to fully understand the safe use of Multiphor Il Electrophoresis System WARNING be followed to avoid personal injury It is important not to proceed until all stated conditions are met and clearly understood f The WARNING sign highlights instructions that must CAUTION The Caution sign highlights instructions that must be followed to avoid damage to the product or other equipment It is important not to proceed until all stated conditions are met and clearly understood Note The Note sign is used to indicate information important for trouble free and optimal use of the product CE Certifying This product meets the requirements of applicable CE directives A copy of the corresponding Declaration of Conformity is available on request The CE symbol and corresponding declaration of conformity is valid for the instrument when it is used as a stand alone unit or connected to other CE marked GE Healthcare instruments or connected to other products recommended or described in this manual and used in the same state as it was delivered from GE Healthcare except for alterations described in this manual WARNING This is a Class A product In a domestic environment this product may cause radio interference in which case the user may be required to take adeq
25. GE SDS and Native PAGE IEF Kit 18 1102 45 0 5 x 125 x 260mm Gradient Maker 18 1013 72 homogeneous and gradient gel ExcelGel SDS Buffer Strip Positioner 80 6442 90 IEF in polyacrylamide SDS and Native PAGE IEF Kit 18 1102 45 0 5 x 125 x 260mm 2 D first dimension Immobiline Immobiline DryStrip Kit 18 1004 30 DryStrip Reswelling Tray 7 18 cm 80 6371 84 Reswelling Tray 7 24 cm 80 6465 32 Electrophoretic transfer NovaBlot Kit 18 1016 86 FilmRemover 18 1013 75 This User Manual is comprised of the following sections L Introduction includes a general description of Multiphor Il system and dedicated precast gels a guide to the application kits and the manual structure Description of parts describes in detail the components of Multiphor Il Electrophoresis Unit Installation contains a detailed description of how to install Multiphor Il Electrophoresis Unit and Multiphor Il NovaBlot Unit Operation contains information on the operating procedure for SDS and native polyacrylamide gel electrophoresis isoelectric focusing 2 D electrophoresis and electrophoretic transfer Maintenance gives cleaning recommendations to help you maintain your Multiphor Il unit Trouble shooting offers suggestions for correcting problems that may occur Multiphor Il application kits and accessories describes in detail the contents assembly and use of each Multiphor Il application kit Ordering information
26. Product Basic configuration Multiphor Il Electrophoresis Unit Application kits and accessories SDS and Native PAGE IEF Kit Immobiline DryStrip Reswelling Tray for 7 18 cm IPG strips Immobiline DryStrip Reswelling Tray for 7 24 cm IPG strips Immobiline DryStrip Kit for running 1 to 12 Immobiline DryStrip gels for use with Multiphor Il only NovaBlot Kit for electrophoretic transfer Multiphor Il Buffer Strip Positioner complete FlexiClamps FilmRemover for removing plastic gel backing before electrophoretic transfer Gradient Maker 100 ml Roller Template 125x260 mm Lever and Wire Assemblies for Film Remover Levelling Feet Cooling plates Cooling Plate ceramic 210 x 270 mm Grommets Cooling Tubing 8 12 mm Tubing Connector Set female and male Insulation for Cooling Tubing 14 27 mm Anade and Cathode Electrode Leads for 18 1004 31 Immobline DryStrip Kit Tray Quantity 10 Code No 18 1018 06 18 1102 45 80 6371 64 80 6465 32 18 1004 30 18 1016 86 80 6442 90 18 1013 73 18 1013 75 80 6196 09 80 1106 79 80 1129 55 18 1013 79 18 1026 40 18 1103 46 80 1106 58 80 1106 56 18 1104 26 80 1116 11 18 103 7 44 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 67 10 Ordering information Product Quantity Code No Electrodes and electrode holders 68 EPH IEF Electrode anode 1 18 1121 53 EPH IEF Electrode cathod
27. SDS Gradient 8 18 Running conditions Phase Voltage Current Power Time Temp V MA W min C Run 600 50 30 i 15 Or until the Bromophenol Blue front reaches the anode buffer strip ExcelGel XL SDS Gradient 12 14 Running conditions Phase Voltage Current Power Time Temp V MA W min C Run 1000 40 40 103 15 Or until the Bromophenol Blue front reaches the anode buffer strip ExcelGel SDS Homogeneous 7 5 12 5 and 15 Running conditions ExcelGel SDS Voltage Current Power Time Temp Homogeneous V mA W min ye 7 5 and 12 5 600 50 30 80 IS 15 600 30 30 140 15 Or until the Bromophenol Blue front reaches the anode buffer strip Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 43 5 Operation 44 Ampholine PAGplate for IEF Running conditions for Ampholine PAGplate pH range Voltage Current Power Time Temp V mA W h C 3 5 9 5 1500 50 30 1 5 10 4 0 6 5 2 000 25 25 2 95 10 5 5 8 5 1 600 50 25 203 10 4 0 5 0 1 400 50 30 3 0 10 5 0 6 5 2 000 15 20 3 0 10 If half a gel is used halve the current and power settings CleanGel IEF 3 10 Running conditions for one CleanGel IEF 3 10 Phase Voltage Current Power Time Temp V MA W min e Prefocusing 700 12 8 20 10 Sample entry 500 8 8 20 10 Isoelectric focusing 2000 14 14 90 10 Band sharpening 2500 14 18 10 10 If half a gel is used halve the current and power settings 2 D electrophor
28. Voltage Current Power Time Temp V MA W min C 1 600 20 30 25 30 15 e 600 50 30 DRS 15 3 600 50 30 Qe ales When the Bromophenol Blue dye front has moved 4 6 mm for ExcelGel XL SDS gradient 12 14 and 1 2 mm for ExcelGel SDS gradient 8 18 from Immobiline DryStrip remove the strip and the application pieces When the front has moved a further 2 mm move the cathodic buffer strip forward to cover the area of removed Immobiline DryStrip by 1 2 mm Adjust the position of the cathodic electrode When the Bromophenol Blue front has just reached the anodic buffer strip electrophoresis is continued for 5 min and should then be stopped Remove the buffer strips Further information about the gels and running conditions are supplied with the products Running conditions for ExcelGel 2 D Homogeneus 12 5 Phase Voltage Current Power Duration V mA W h min 1 600 20 30 0 351 2 600 50 30 LS Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Maintenance 6 6 Maintenance WARNING Remove liquid or dirt from the system surface using a cloth and if necessary a mild cleaning agent WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system WARNING When using hazardous chemicals
29. a Tel 01 57606 1619 Fax 01 57606 1627 e Belgium Tel 0800 73 888 Fax 03 272 1637 e Canada Tel 800 463 5800 Fax 800 567 1008 Central East amp South East Europe Tel 43 1 982 3826 Fax 43 1985 8327 e Denmark Tel 45 16 2400 Fax 45 16 2424 e Finland amp Baltics Tel 358 0 9 512 39 40 Fax 358 0 9 512 39 439 e France Tel 01 69 35 67 00 Fax 01 69 41 96 77 e Germany Tel 0761 4903 490 Fax 0761 4903 405 e Italy Tel 02 27322 1 Fax 02 27302 212 Japan Tel 81 3 5331 9336 Fax 81 3 5331 9370 e Latin America Tel 55 11 3933 7300 Fax 55 11 3933 7304 e Middle East amp Africa Tel 30 210 9600 687 Fax 30 210 9600 693 e Netherlands Tel 0165 580 410 Fax 0165 580 401 e Norway Tel 815 65 555 Fax 815 65 666 e Portugal Tel 21 417 7035 Fax 21 417 3184 e Russia other C 1 S amp N I S Tel 7 095 232 0250 956 1137 Fax 7 095 230 6377 e South East Asia Tel 60 3 8024 2080 Fax 60 3 8024 2090 e Spain Tel 93 594 49 50 Fax 93 594 49 55 e Sweden Tel 018 612 1900 Fax 018 612 1910 e Switzerland Tel 0848 8028 12 Fax 0848 8028 13 UK Tel 0800 616928 Fax 0800 616927 e USA Tel 800 526 3593 Fax 877 295 8102 imagination at work User Manual 18 1103 43 AK 02 2006 E Elanders sterv la 2006
30. anual 18 1103 43 Edition AK 2I 5 Operation 28 5 3 Isoelectric focusing using Ampholine PAGplate This section describes the running procedure for IEF using Ampholine PAGplate For further information see the instructions supplied with the product Switch on MultiTemp Il thermostatic circulator and set the temperature normally 10 C for polyacrylamide IEF 15 minutes before starting the experiment Cover the holes in the safety lid with tape to limit the amount of CO in contact with the gel thereby improving the basic region of the pH gradient If desired 100 ml of 1 M sodium hydroxide solution may be poured into the buffer chambers to absorb CO and further improve gradient stability Make up about 100 mls of the required electrode solutions See Table Electrode solutions for IEF using Ampholine PAGplate pH range Anode solution Cathode solution 5 995 1 M Phosphoric acid 1 0 M NaOH 4 0 6 5 0 1 M Glutamic acid 0 1 M B alanine 5 5 8 5 0 4 M HEPES 0 1 M NaOH 4 0 5 0 1 M Phosphoric acid 1 0 M Glycine 5 0 6 5 0 01 M Acetic acid 0 01 M NaOH Note Wear clean gloves when working with polyacrylamide gels Pipette 1 ml of insulating fluid kerosene or light paraffin oil onto the cooling plate Open one package of Ampholine PAGplate and position the gel with the stiff plastic film facing down on the cooling plate Make sure no air bubbles are trapped under the gel Use the screened template on the cooling plate to centre the gel
31. ation strips for up to 40 ul of sample in 26 slots IEF SDS application strips for up to 20 ul of sample in 52 slots Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Immobiline applicator strip for up to 5 ul of sample in 52 slots Immobiline applicator strip is designed to counteract lateral band spreading Sample application pieces hold approximately 20 ul of sample For smaller volumes cut the paper pieces to an appropriate size At least 24 application pieces size 5 x 10 mm and 50 application pieces size 2 5 x 5 mm can be placed on one gel Apply the sample about 1 cm away from the cathodic buffer strip and 1 cm away from each short side of the gel For the best results remove the application pieces 15 min after electrophoresis has started ExcelGel SDS ExcelGel Homogeneous 7 5 12 5 15 and CleanGel are available with various numbers of sample wells for various volumes Samples are applied directly into the wells immediately prior to the run Electrophoresis Connect Multiphor Il to MultiTemp Il thermostatic circulator Switch on MultiTemp Il 15 minutes before starting the experiment and set the temperature to 15 C Always wear clean gloves when working with polyacrulamide gels and buffer strips particularly when using sensitive staining methods Remove one ExcelGel SDS from the package Pipette 1 ml of insulating fluid kerosene or light paraffin oil onto the cooling plate Place the gel with
32. cast gels Running procedures for electrophoretic transfer are also included For laboratory cast gels use the running conditions recommended in Electrophoresis in Practice A Guide to Theory and Practice by Reiner Westermeler Multiphor Il contains two alternative electrode configurations EPH IEF electrodes for use with buffer strips or electrode strips EPH electrodes for use with electrode wicks and buffer chambers IEF SDS PAGE and native PAGE are most conveniently performed using EPH IEF electrodes and buffer strips The strips are applied on the gel edges with the electrodes on top ExcelGel Electrode wicks The buffer chambers are located below the cooling plate with the electrodes immersed in buffer solution Paper wicks connect the buffer solution with the gel This method is used for SDS PAGE and native PAGE The optional card mounted EPH electrodes 18 1122 19 and 18 1122 20 for electrophoresis using buffer chambers across the width of the cooling plate are moulded from polypropylene and support the platinum wire The anode cable red and cathode cable black carry female pin connectors for attachment to the male pins at the front of the buffer tank Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 19 5 Operation 20 5 1 Electrophoresis using ExcelGel SDS and buffer strips This section describes the running procedure for SDS PAGE usin
33. commended No cooling is necessary since negligible heat is produced during the transfer Immobilizing membranes The immobilizing membrane is an important factor affecting the efficiency of the electrophoretic transfer The most important requirements for an immobilizing membrane are e High binding capacity for the molecules of interest e Preservation of the biologic activity of the molecules of interest e No interference with subsequent detection methods e Chemical and mechanical stability to assay conditions e Provision of an accurate reflection of the original separation Nitrocellulose membranes are the standard medium for electrophoretic transfer of proteins and nucleic acids This is due to their high binding capacity versatility and easy use Nitrocellulose membranes are available in various pore sizes 0 45 um is most commonly used however low molecular proteins may be lost By using pore sizes of 0 2 or 0 1 um most proteins are retained Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 35 5 Operation 36 Nitrocellulose membranes can be probed several times The membranes require no activation and the functional groups have an extended lifetime Protein patterns on nitrocellulose membranes can be easily detected with most conventional stains as well as by autoradiographic immunoenzymatic and fluorescent methods Other membranes are nylon based membranes diazobenzyloxymethyl DBM and diazophenul
34. de solution Draw the gel solution into a syringe or a graduated pipette Fill the mould checking that air bubbles are not trapped along the rubber U frame or around the slots When casting a gradient gel position the mould horizontally using the Levelling Set and place the Gradient Maker as illustrated Lay the end of the tubing from the Gradient Maker against the 1 mm glass plate or GelBond PAGfilm The slot former will otherwise disturb the flow of the solution To open the 1mm glass plate mould remove the four FlexiClamps Carefully insert one or two thin bladed spatulas between the gel surface and slot former on one of the short sides Twist gently in order to introduce air across the whole of the short side Twist more firmly to slowly separate the U frame from the gel surface Remove the U frame Carefully remove any unpolymerized acrylamide from the edge of the gel with a paper tissue Separate the gel support from the thick glass plate The gel is now ready to use To open the mould including GelBond PAGfilm remove the four FlexiClamps and insert the spatula between the 3 mm thick glass plate and film Remove the glass plate and dry the back of the film Turn the mould upside down with the glass plate with U frame on top and gently peel the film with gel away from the glass Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Multiphor Il application kits and accessories 9 9 2 Buffer Strip Positioner The Mu
35. e 1 18 1121 52 Electrode Holder for 18 1106 60 61 1 80 1106 55 EPH Electrode long anode 1 18 1122 20 EPH Electrode long cathode 1 18 1122 19 Electrode Immobiline DryStrip Kit anode 1 18 1018 66 Electrode Immobiline DryStrip Kit cathode 1 18 1018 67 Tray and Electrode Holder for 18 1018 66 67 1 18 1004 31 NovaBlot Electrode anode 1 80 1257 87 NovaBlot Electrode cathode 1 18 1019 86 Glass plates and trays 125 x 260 x 3 mm 2 80 1106 99 125 x 260 x 1 mm 15 80 1106 29 125 x 260 mm 0 5 mm U frame 2 80 1106 89 125 x 260 mm 1 0 mm U frame 2 80 1106 91 125 x 260 mm 2 0 mm U frame 2 80 1106 92 Glass plate treatment Bind Silane 100 ml 17 1330 01 Repel Silane 500 ml 17 1331 01 Gel support GelBond PAGfilm 124 x 258 mm 50 80 1129 36 Paper electrode strip and wicks IEF Electrode Strip 100 18 1004 40 EPH Electrode Wick 82 x 130 mm 500 80 1129 53 EPH Electrode Wick 104 x 253 mm also used as PEGG print paper and with agarose IEF 500 80 1129 52 Electrode Paper NovaBlot 200 x 250 mm 500 80 1106 19 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Ordering information 10 Product Quantity Code No Sample application SDS Sample Application Strip 26 samples 40 ul 5 18 1002 74 IEF SDS Sample Application Strip 52 samples 5 20 ul 5 18 1002 26 IEF Sample Application Pieces 200 80 1129 46 Sample Cups Immobiline DryStrip Kit 60 18 1004 35 Preserving C
36. e holder from the unit Carefully remove the gel and proceed with staining or preparation for the second dimension as required WARNING Always turn OFF the power supply before opening the safety lid Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 31 5 Operation 32 Detection methods For automated silver and coomassie staining of polyacrylamaida gels see Protocol guide Hoefer Automated Gel Stainer 80 6343 34 PhastGel Blue staining On completion of isoelectric focusing remove the electrode strips from the gel IMmerse the gel in 250 ml of solution according to the schedule below See section 4 9 for stock solutions The staining and destaining steps should be carried out on a shaking table Step No Solution Time min Temp C 1 Fixing solution N 20 25 2 Destaining solution 2 23 3 Staining solution K 10 60 4 Destaining solution 20 23 5 Destaining solution 30 23 6 Preserving solution O 10 23 The staining solution should be heated to 60 C and poured over the gel No further heating is necessary Destain the gel using several changes of destaining solution 1 until the background is clear Change the solution frequently particularly at the beginning in order to speed up the destaining To preserve the gel soak a cellophane sheet in preserving solution I and lay it on the gel surface Remove any air bubbles and wrap the excess cellophane around the glass plate An additional glass
37. e socket of the cathode electrode to the pin at the front of the unit and the anode electrode pin to the socket at the back Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Place the safety lid in position by matching the extensions on the back of the lid with the openings on the base unit Using the extensions at the back as a hinge connect the male and female banana plugs by pressing down firmly on the front of the lid Running conditions Connect Multiphor Il to the power supply Set the running conditions given in the table below Start the isoelectric focusing by turning on the power supply Observe the time limits closely If the isoelectric focusing is run too long the pH gradient will begin to drift towards the cathode Suggested running conditions for Ampholine PAGplate pH range Voltage V Current mA Power W Time h 3 5 9 5 1500 50 30 LS 4 0 6 5 2 000 25 25 O 5 5 8 5 1 600 50 25 2 5 4 0 5 0 1 400 50 30 3 0 5 0 6 5 2 000 15 20 3 0 Note If only half a gel is used halve the current and power settings Remove the sample application pieces with forceps after approximately half the focusing time has expired When a sample application strip is used let it remain on the gel during the focusing but remove it before placing the gel in the fixing solution Ending the run Turn off the power supply and disconnect Multiphor Il from the power supply Remove the safety lid and electrod
38. each 17 1342 01 IEF CleanGel IEF 5 18 1035 32 GelPool for gel rehydration 1 18 1031 58 PaperPool for electrode strips I 18 1031 59 Ampholine PAGplate 5 80 1124 80 pH 3 5 9 5 Ampholine PAGplate 5 80 1124 81 pH 4 0 6 5 Ampholine PAGplate 5 80 1124 82 pH 5 5 8 5 Ampholine PAGplate 5 80 1124 83 pH 4 0 5 0 Immobiline DryStrip Gels Immobiline DryStrip pH 3 5 4 5 24 cm 12 17 6002 38 Immobiline DryStrip pH 4 0 5 0 24 cm 12 17 6002 39 Immobiline DryStrip pH 4 5 5 5 24 cm 12 17 6002 40 Immobiline DryStrip pH 5 0 6 0 24 cm 12 17 6002 41 Immobiline DryStrip pH 5 5 6 7 24 cm 12 17 6002 42 Immobiline DryStrip pH 6 9 24 cm la 17 6002 47 Immobiline DryStrip pH 3 7 NL 24 cm 12 17 6002 43 Immobiline DryStrip pH 3 10 24 cm 12 17 6002 44 Immobiline DryStrip pH 3 10 NL 24 cm 12 17 6002 45 Immobiline DryStrip pH 4 7 24 cm 12 17 6002 46 Immobiline DryStrip pH 3 5 4 5 18 cm l2 17 6001 83 Immobiline DryStrip pH 4 0 5 0 18 cm 12 17 6001 84 Immobiline DryStrip pH 4 5 5 5 18 cm 12 17 6001 85 Immobiline DryStrip pH 5 0 6 0 18 cm 12 17 6001 86 Immobiline DryStrip pH 5 5 6 7 18 cm 12 17 6001 87 Immobiline DryStrip pH 4 7 18 cm 12 17 1233 01 Immobiline DryStrip pH 6 9 18 cm 12 17 6001 88 Immobiline DryStrip pH 6 11 18 cm 12 17 6001 97 Immobiline DryStrip pH 3 10 NL 18 cm 12 17 1235 01 Immobiline DryStrip pH 3 10 18 cm 12 17 1234 01 Immobiline DryStrip pH 4 7 13 cm 12 17 6001 13 Immobiline DryStrip pH 6 11 13 cm 12 17 6001 96 Immobiline DryStr
39. ed for connection to power supplies ensure safe operation at high voltages The polycarbonate lid snugly fits the contours of the buffer tank This makes it possible to reduce the atmospheric CO content around the gel important for IEF at basic pH intervals and provides increased protection against condensation Note Polycarbonate is not resistant to concentrated acids and bases or to halogenated and aromatic hydrocarbons Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Description of parts 3 The ceramic aluminium oxide cooling plate measures 210 x 270 mm supports the gel and provides uniform temperature control Aluminium oxide is an excellent heat conductor and electrical insulating material t WARNING The cooling plate is rated for operation at up to 3 5 kV p p To facilitate the correct positioning of electrophoresis gels the surface of the cooling plate is screened with a template measuring 190 x 250 mm The two grommets are connected to the inlet and outlet tubes of the cooling plate which can then be connected to a thermostatic ciculator such as MultiTemp III WARNING Only use water with high electrical resistance as coolant and NEVER EXCEED maximum pressure of 0 5 bar The cooling tubing tubing connector set and hose clamps provide a flexible and safe way to connect Multiphor Il to a thermostatic circulator such as MultiTemp III Po ai The electrode holde
40. ellophane Sheets 210 x 320 mm 50 80 1129 38 Mylar Sheets 125 x 260 mm 5080 1129 39 Membranes electrophoretic transfer Nitrocellulose 0 20 um 150x200 mm 15 80 1098 91 Nitrocellulose 0 45 um 150x200 mm 15 80 1098 90 ProBind 45 NC 0 45 um roll 0 2x3 0 m 1 80 1247 86 GeneBind 45 nylon 0 45 um roll 0 2x3 0 m 80 1247 87 Tapes Dymo 0 25x9 mm 3 m 1 80 1129 50 10 2 MultiTemp Ill Product Quantity Code No MultiTemp III 1 18 1102 77 thermostatic circulator 100 120 V MultiTemp III 1 18 1102 78 thermostatic circulator 220 220 V Cooling Tubing 8 12 mm 4m 80 1106 56 Tubing Connector Set female and male 4 18 1104 26 Insulation for Cooling Tubing 14 27 mm 2m 80 1116 11 3 way Valve Set 1 18 1106 39 10 3 EPS Power Supplies Product Quantity Code No EPS 3501 XL 1 18 1130 05 35 3500 V 1 400 mA EPS 3501 1 18 1130 04 35 5500 V 1 150 mA EPS 1001 1 18 1130 03 5 1000 V 1 400 mA EPS 601 1 18 1130 02 6 600 V 1 400 mA EPS 01 1 18 1130 01 58 300 V 1 400 MA Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK 69 10 Ordering information 70 10 4 Precast gels and buffer strips Product Quantity Code No SDS PAGE and Native PAGE ExcelGel SDS Homogeneous 7 5 6 80 1260 01 ExcelGel SDS Homogeneous 12 5 6 80 1261 01 ExcelGel SDS Homogeneous 15 6 80 1262 01 ExcelGel SDS gradient 8 18 6 80 1255 53 ExcelGel XL SDS gradient 12 14 3 17 1236 01 ExcelGel SDS Buffer Strips anode and cathode 6
41. esis using Immobiline DryStrip and ExcelGel SDS First dimension Option 1 EPS 3500 XL Power Supply using a voltage gradient The parameters below may be used for up to 12 strips Programme for Immobiline DryStrip pH 3 10 110 mm All steps are run at 10 C Phase Voltage V 1 300 2 300 3 2000 4 2000 Current MA 1 1 1 1 Power W 5 5 5 5 Time h 0 1 45 5 6 5 16 Vh 1 1350 5 50 13000 20100 Programme for Immobiline DryStrip pH 3 10 L 180 mm All steps are run at 10 C Phase Voltage Current Power Time Vh V MA W h 1 500 1 5 0 1 1 2 500 1 5 3 1500 3 3500 1 5 3 10000 4 3500 1 5 123 43750 Total 20 5 55250 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Programme for Immobiline DryStrip pH 3 10 NL 180 mm All steps are run at 10 C Phase Voltage Current Power Time Vh V MA W h 1 500 1 5 0 1 1 2 500 1 5 s 2500 E 3500 1 5 5 10000 4 3500 1 5 9 5 32400 Total 19 5 44900 Programme for Immobiline DryStrip pH 4 7 110 mm All steps are run at 10 C Phase Voltage Current Power Time Vh V MA W h 1 300 1 5 0 1 1 2 300 1 3 6 1800 E 3500 1 5 5 9500 4 3500 1 5 55 19250 Total 16 5 30550 Programme for Immobiline DryStrip pH 4 7 180 mm All steps are run at 10 C Phase Voltage Current Power Time Vh V MA W h 1 500 1 5 0 1 1 2 500 1 5 1 500 5 3500 1 3 5 10000 4 3500 1 5 10 350
42. g buffer strips The running of ExcelGel SDS gradient 8 18 using ExcelGel SDS buffer strips is chosen as an example but the basic method is applicable to all SDS PAGE and native PAGE gels ExcelGel SDS gradient 8 18 is a 0 5 mm thin precast polyacrylamide gel for horizontal electrophoresis of SDS denatured proteins To facilitate handling the gel is cast on a plastic support During the run the precast SDS buffer strips supply the gel with buffer ions For further information see the information supplied with ExcelGel SDS gels Sample preparation Dissolve the samples in sample buffer B for recipes see Section 4 9 Stock solutions Then heat the sample solution at 95 C for 3 minutes The sensitivity of your development technique and the volume of sample applied to the gel will determine the lower limit of your sample concentration Generally the sample must contain 200 to 500 ng of each component for Coomassie staining and at least 10 25 ng of each component for silver staining For molecular weight determination we recommend the use of molecular weight calibration kits LMW and HMW SDS Sample application In horizontal electrophoresis there are three methods of applying the sample application strips paper pieces and sample wells Sample application strips are put on the gel surface forming sample slots Silicone rubber sample application strips are specially al The following application strips are available SDS applic
43. he table below All steps should be carried out at room temperature in daylight while gently shaking the solution Use 250 ml of solution for each step See section 4 9 for stock solutions Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Time schedule for silver staining Step No Solution Time min i Fixing solution C 30 2 Incubation solution D 30 3 Distilled water 34 4 Silver solution E 40 5 Developing solution F 5 15 6 Stop solution G 10 7 Distilled water 3X5 8 Preserving solution L 20 Short development times will give a lightly stained gel Long development times will give a dark gel To preserve the gel soak a cellophane sheet in preserving solution L and lay it on the gel surface Remove any air bubbles and wrap the excess around the glass plate An additional glass plate may be placed underneath during drying to stop the cellophane from shifting or wrinkling Leave the gel at room temperature until it is completely dry 5 2 Electrophoresis using buffer chambers This section describes the running procedure when using buffer chambers To place the electrodes in the buffer chamber remove the cooling plate from the buffer tank To reduce the effect of electrolysis products during the electrophoresis the electrodes should be positioned as far as possible from the wicks Therefore when performing electrophoresis across the large cooling plate the electrodes should be placed
44. ies GE imagination at work and GE monogram are trademarks of General Electric Company GE Healthcare Bio Sciences AB Hoefer is a trademark of Hoefer Inc Triton is a trademark of Bj rkgata N 3 0 Union Carbide Chemicals and Plastics Co Tween is a trademark of 751 84 U ppsala ICI Americas Inc Rynite is a trademark of DuPont Company Sweden All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them GE Healthcare reserves the right subject to any regulatory and contractual approval if required to make changes in specifications and features shown herein or discontinue the product described at any time without notice or obligation Contact your local GE Healthcare representative for the most current information 2006 General Electric Company All rights reserved GE Healthcare Bio Sciences AB a General Electric Company GE Healthcare Bio Sciences AB Bjorkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare UK Ltd Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan Asia Pacific Tel 852 2811 8693 Fax 852 2811 5251 e Australasia Tel 61 2 9899 0999 Fax 61 2 9899 7511 e Austri
45. ing Burning at slots or Polypeptide complexes are too big to accumulation of enter the gel and cause water in the slots electroendosmosis Trouble shooting guide to IEF Symptom Cause Current increases Electrode strips applied incorrectly with time in relation to electrode polarity Cathode and anode polarities reversed Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Remedy Check the safety plug insertion Check the pin and socket connections Connect the bridging contact Press firmly on the safety lid Lower the electrode holder so that the electrodes are in contact with the electrode strips Check that the electrodes are clean and intact and sit in the centre of the isoelectric focusing strips over the entire length Check the contact Check the cooling If SDS PAGE add DTT once again and boil the sample Remedy Check the electrode polarity and the pH of the electrode strips Check the pin and socket connections the gel orientation and the pH of the applied electrode strips 51 8 Trouble shooting Symptom Sparking on the gel Water droplets on gel Drying out of the gel near the electrodes Sparking along edge of gel onto cooling plate Condensation over the entire surface of the glass electrode holder Local condensation on the glass electrode holder Excessive amount of condensation along electrode strips 52 Cause Gel dried out insufficient cooling Excessi
46. ip pH 3 10 NL 13 cm 12 17 6001 15 Immobiline DryStrip pH 3 10 13 cm 12 17 6001 14 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Ordering information 10 Product Quantity Code No Immobiline DryStrip pH 4 7 11 cm 12 18 1016 60 Immobiline DryStrip pH 6 11 11 cm 12 17 6001 10 Immobiline DryStrip pH 3 10 11 cm 12 18 1016 61 Immobiline DryStrip pH 4 7 7 cm 12 17 6001 10 Immobiline DryStrip pH 6 11 7 cm 12 17 6001 94 Immobiline DryStrip pH 3 10 NL 7 cm be 17 6001 12 Immobiline DryStrip pH 4 7 7 cm e 17 6001 10 Immobiline DryStrip pH 3 10 7 cm 12 17 6001 11 IPG Buffer IPG Buffer pH 3 5 5 0T 1 mi 17 6002 02 IPG Buffer pH 4 5 5 5 1 mi 17 6002 04 IPG Buffer pH 5 0 6 0 1 mi 17 6002 05 IPG Buffer pH 5 5 6 7 1 mi 17 6002 06 IPG Buffer pH 4 78 1 mi 17 6000 86 IPG Buffer pH 6 117 1 mi 17 6001 78 IPG Buffer pH 3 10 NL 1 17 6000 88 IPG Buffer pH 3 10 1 mi 17 6000 87 NL increased resolution between pH 5 7 t Use IPG Buffer pH 3 5 5 0 for pH 3 5 4 5 and 4 0 5 0 IPG strips t Use IPG Buffer pH 6 9 and pH 6 11 IPG strips 8 Use IPG Buffer pH 4 7 for pH 3 7 IPG strips Product Quantity Code No Second dimension ExcelGel 2 D Homogeneus 17 6002 21 ExcelGel SDS gradient 8 18 80 1255 53 110 x 245 x 0 5 mm ExcelGel XL SDS gradient 12 14 3 17 1236 01 180 x 245 x 0 5 mm ExcelGel SDS Buffer Strips anode and cathode 6 each 17 1342 01 10 5 Molecular weight and pl markers Product Quantity Code No MW range 2 51
47. ltiphor Il Buffer Strip Positioner is a frame with slots thats sits on top of an ExcelGel SDS gel on the Multiphor Il cooling plate Theslots in the positioner facilitate placement of the buffer strips for electrophoresis and hold them securely in place A locking cam secures the positioner on the cooling plate Cathode Locking ca Anode Fig 1 Features of the Multiphor Il Buffer Strip Positioner Slot Use for placing 1 Cathodic buffer strip Phase 1 or entire ruun Z Sample wells Immobiline DryStrip gels IPG strips Phase 1 Cathodic buffer strip Phase 2 3 Anodic buffer strip with 11 x 25 cm ExcelGel SDS gels 4 Anodic buffer strip with 18 x 25 cm ExcelGel SDS gels Designation Code No Multiphor Il Buffer Strip Positioner 80 6442 90 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 59 9 Multiphor ll application kits and accessories 9 3 Immobiline DryStrip Reswelling Try The Immobiline DryStrip Reswelling Trays have twelve independent reservoir slots that can each hold a single IPG strip Separate slots allow the rehydration of individual IPG strips in a minimal volume of solution Designation Code No Immobiline DryStrip Reswelling Tray 80 6371 84 for 7 18 cm IPG Strips Immobiline DryStrip Reswelling Tray 80 6465 32 for 7 24 cm IPG Strips 9 4 Immobiline DryStrip Kit This kit is used for running IEF with Immobiline DryStrip for the first dimension in 2 D electrophoresis Twelve strip
48. make sure that the entire system has been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water before service and maintenance A few standard measures are necessary to keep Multiphor II in full functioning order After isoelectric focusing remove the electrodes from the electrode holder and rinse with distilled water to remove the strong acidic and basic solutions Do not submerge the cable containing the pin or socket Air dry or carefully dry with paper tissue Check that the platinum wire is not damaged After electrophoresis using the buffer chambers remove the electrodes Rinse them in distilled water and air dry Take care not to damage the platinum electrodes Rinse the buffer chambers with distilled water between buffer changes and after use Do not immerse the socket connector Air dry or carefully dry with a paper towel Following electrophoretic transfer remove all remaining filter papers from the NovaBlot unit Remove the anode and cathode plates and rinse them in distilled water Do not immerse the electrode leads in water Leave to air dry For longer life of NovaBlot electrodes store them either bu 1 Placing 3 cm thick plastic foam between the electrodes as if for transfer or 2 Store the electrodes on backs without foam sandwiched between 6 1 Recycling This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste
49. mation 10 1 MOIO iii 67 10 2 LU VO ARR RR 69 10 EPSPONCCSUP Sura 69 104 Preca gel dnd PU NSF Picci 70 10 5 Molecular weight and pl MarkeFS ccocncconionucinucinonoconacnononsncencnnnoncncesss 71 10 6 Carrier GIP MONG COS escritas 71 10 7 PlusOne electrophoresis ChEMICA S iii 73 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 5 Contents 6 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Introduction 1 1 Introduction Multiphor M Il electrophoresis system is a versatile modular system for horizontal electrophoresis isoelectric focusing 2 D electrophoresis and electrophoretic transfer For ease of use and reproducible results an innovative range of precast gels for all major electrophoretic techniques is available with Multiphor Il Technique Precast Gel SDS and Native PAGE ExcelGel SDS Gradient ExcelGel SDS Homogeneous IEF Ampholine PAGplate CleanGel IEF 2 D electrophoresis Immobiline DryStrip ExcelGel SDS gradient Homogeanus If laboratory cast gels are preferred an application kit and accessories can be added to the basic electrophoresis unit Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Pi 1 Introduction The following guide summarizes how you can expand and use Multiphor Electrophoresis Unit with application kits and accessories Application Recommended Kit Accessory Code No SDS and Native PA
50. nd gently push out the bubbles r 6 Immerse nine filter papers in cathode solution T Place these filter papers on top of the gel to complete the transfer sandwich oj Filter paper 6 Dialysis membrane ra Filter paper cathodic 3 _ 7 Slab gel _Transfer N ua sandwich ani membrane Filter paper 6 7 Several gels of the same type and size can be transferred simultaneously Two transfer sandwiches can be put on top of each other The cellophane dialysis membrane placed between each transfer sandwich prevents crosscontamination between transfer sandwiches The maximum gel size is 200 x 250 mm If small gels 125 x 250 mm are to be transferred NovaBlot will accept up to four gels for simultaneous transfer by assembling two transfer sandwich stacks side by side Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 39 5 Operation 40 To ensure that the current passes through the gel all components of the transfer sandwich are cut to the same size as the gels to be transferred 8 Saturate the cathode electrode plate with distilled water and remove any excess with absorbent paper Place the cathode on top of the transfer sandwich and connect the socket on the black cathode lead to the cathode pin in the Multiphor Il base 9 Close the Multiphor Il safety lid and connect the unit to the power supply It is recommended to run the transfer at a constant current of 0 8 m
51. ol IEF SDS PAGE 59 F 17 1318 02 Mercaptoethanol IEF SDS PAGE 25 ml B 17 1317 01 Glycerol 87 IEF PAGE Sequencing 1000 ml A 17 1325 01 Detergents Sodium dodeculsulphate PAGE 100 g A 17 1313 01 Triton X 100 IEF PAGE Sequencing 500 ml G 17 1315 01 CHAPS IEF PAGE Sequencing 1g F 17 1314 01 Tween 20 IEF PAGE Sequencing 500 ml G 17 1316 01 Stains Silver Staining Kit Protein Protein detection For 10 20 gels D 17 1150 01 Silver Staining Kit DNA Nucleic and detection For 10 20 gels D 17 6000 30 Ethidium bromide solution 10 mg ml DNA RNA detection 10 ml A 17 1328 01 Bromophenol Blue IEF PAGE Sequencing 10g A 17 1329 01 Glass plate treatment Repel Silane ES IEF PAGE Sequencing 500 ml C 17 1331 01 Bind Silane IEF PAGE Sequencing 25ml C 17 1330 01 Others DryStrip Cover Fluid 2 D Immobiline DryStrip 1000 ml G 17 1335 01 Amberlite IRN 150L Purifying solutions 500 g A 17 1326 01 Storage A room temp B dry at room temp C dry amp dark at room temp D dark at 4 C to 8 C E dry amp dark at 4 C to 8 C F dry at 4 to 8 C G dark at room temp Store well sealed 1 Add 100 ml 2 Add 500 ml Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 10 Ordering information 74 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK www gehealth care com Multiphor Immobiline CleanGel Ampholine MultiTemp ExcelGel Pharmalyte and NovaBlot are trademarks of GE Healthcare compan
52. otein staining identification of specific antigens or antibodies immunoblotting and glycoprotein detection By using different electrode solutions and running conditions it is possible to transfer proteins from SDS PAGE native PAGE agarose gels and isoelectric focusing gels The speed and efficiency of the electrophoretic transfer using NovaBlot system is dependant on e Characteristics of the immobilizing membrane e Characteristics of the transfer buffer e Molecular weight and charge of the protein e Gel thickness and concentration of acrylamide and bisacrylamide e Voltage current and transfer time The semi dry transfer technique uses filter papers soaked in buffer as the only buffer reservoir Both discontinuous and continuous buffer systems can be used in the filter paper layers Methanol in the buffer solution prevents swelling of polyacrylamide gels However it may have the disadvantage of denaturing or fixing the proteins in the gel resulting in a lower transfer efficiency Conversely methanol may increase the protein binding capacity of the nitrocellulose membrane by strengthening the hydrophobic interactions The transfer speed and efficiency can also be increased by increasing the protein charge i e adding 0 05 SDS in the transfer buffer The transfer is normally finished in about one hour If it is necessary to transfer for more than 1 hour due to unusual sample characteristics rewetting of the cathode filter paper is re
53. r holds the movable EPH IEF electrodes The holder keeps the electrodes away from the gel surface during alignment and then provides a uniform pressure over the buffer strips or electrode strips during the separation Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 13 3 Description of parts 14 The electrode holder consists of a double strength glass plate with ground edges and four corner feet made of RyniteTM FR530 The electrode holder holds one anode and one cathode electrode The EPH IEF electrodes consist of moulded polysulfone bars which support the platinum wire held taut by stainless steel springs The cables are spring reinforced for safetu The anode cable red carries the pin contact to be connected to the socket connector on the buffer tank The cathode cable black carries a female socket connector which fits to the buffer tank pin connector Clamping nuts located at each end of the electrode allow easy adjustment of the electrodes on the holder The distance between the electrodes can be varied from 10 mm to 240 mm Note Polysulfone is not resistant to ketones esters halogenated and aromatic hydrocarbons Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Installation 4 4 Installation For all products check the unassembled parts against the Packing List for the respective product to ensure that all items heve been included For easy connection of Multiphor Il Elec
54. s can be focused simultaneously under a protective layer of silicone oil The high sample capacity allows the application of up to 100 ul on each Immobiline DryStrip Detailed instructions for use are available in the instruction manual provided with this kit sl i de l Kit contents Code No 18 1004 30 Designation Code No Tray and Electrode Holder 18 1004 31 DryStrip Aligner 4 pkg 18 1004 34 DryStrip Kit Electrode cathode 18 1018 67 DryStrip Kit Electrode anode 18 1018 66 Sample Cup Bar 18 1004 33 Sample Cup 6 x 10 pkg 18 1004 35 IEF Electrode Strip 100 pkg 18 1004 40 IEF Sample Application Piece 200 pkg 80 1129 46 Instruction Manual 18 1038 63 Optional accessories Designation Code No Immobiline DryStrip Reswelling Tray 80 6371 84 for 7 18 cm IPG Strips Immobiline DryStrip Reswelling Tray 80 6465 32 for 7 24 cm IPG Strips Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Multiphor ll application kits and accessories 9 9 5 NovaBlot Kit This kit is used for electrophoretic transfer of proteins from polyacrylamide or agarose gels to an immobilizing membrane The maximum gel size is 200 x 250 mm By building transfer sandwiches simultaneous transfer from several gels of the same type can be achieved Up to six transfer sandwiches can be stacked one on top of the other If 125 x 250 mm gels are to be transferred NovaBlot accepts up to six gels for simultaneous transfer by assembling
55. s required If voltage probe measurements are not required remove the isoelectric focusing electrodes from the electrode holder Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Position the empty electrode holder so it lies directly on the electrode wicks This will ensure even contact between the wicks and the gel and stop any moisture from condensing on the gel surface Replace the safety lid on the unit and connect the Multiphor Il unit to the power supply Set the power requirements and start the experiment Typical power settings for agarose immunoelectrophoresis using buffer chambers are constant voltage 20 V cm and current and power set to maximum Run time is 40 60 minutes Bromophenol Blue is used as a tracking due Typical power settings for SDS PAGE electrophoresis using buffer chambers Separation distance cm Voltage Current Power Time Temp V MA W min C 8 600 50 30 100 15 16 1200 50 30 165 15 Ending the run Turn off the power supply Disconnect the Multiphor Il unit from the power supply Take off the safety lid from the unit Carefully remove the electrode holder Gently pull the wicks from the surface of the gel and continue with detection techniques as required WARNING Always TURN OFF the power supply before opening the safety lid Although user safety is not endangered arcing may damage the contacts Multiphor Il Electrophoresis System User M
56. stem User Manual 18 1103 43 Edition AK Multiphor ll application kits and accessories 9 9 7 Roller For use when appluing plastic support films onto glass plates with an interfacing fluid The roller is used to provide even pressure over a large area ensuring adhesion with a minimum amount of fluid and elimination of bubble formation Designation Code No Roller 80 1106 79 Several accessories are available for simple and convenient sample application with Multiphor Il 9 8 Sample application accessories Designation Code No IEF Sample Application Pieces 200 pkg 80 1129 46 The 5 x 10 mm sample application piece made of Paratex can be used for sample volumes in the range 15 20 pl Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 63 9 Multiphor ll application kits and accessories Up to 52 samples can be applied with this strip Each well holds up to 20 ul of sample The applicator strip is made of flexible silicone and is applied directly onto the gel surface Designation Code No SDS Sample Application Strip 26 samples 40 pi 18 1002 74 This strip is recommended for sample application on SDS gradient gels without preformed slots e g ExcelGel SDS gradient 8 18 Up to 26 samples can be applied and each well holds up to 40 ul of sample The strip is made of transparent flexible silicone and is applied directly onto the gel surface Designation Code No IEF SDS Sample Application Strip 52 samples
57. ter 4 Operation Further information can be found in Electrophoresis in Practice Code No 18 1104 12 9 1 SDS and Native PAGE IEF Kit This kit is used for casting 0 5 mm homogeneous or gradient polyacrulamide gels The gels are cast on a 1 mm thick glass plate 125 x 260 mm Alternatively casting can be done on GelBond PAGfilm 124 x 258 mm Optional glass plates with U frames allow casting of 1 0 and 2 0 mm thick gels The kit includes sample application pieces and strips for applying the sample onto the gel surface An optional template and tape allow preparation of a slot former Kit contents Code No 18 1102 45 Designation Code No Glass Plate 125 x 260 0 5 mm U frame 2 pkg 80 1106 89 Glass Plate 125 x 260x1 mm 15 pkg 80 1106 29 Glass Plate 125 x 260x3 mm 2 pkg 80 1106 99 FlexiClamp 6 pkg 18 1013 73 IEF Electrode Strip 100 pkg 18 1004 40 Electrophoresis Wick 104 x 253 mm 500 pkg 80 1129 52 IEF Sample Application Pieces 200 pkg 80 1129 46 IEF SDS Sample Application Strip 52 samples 5 20 pl 5 pkg 18 1002 26 Cellophane Sheets 50 pkg 80 1129 38 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 55 9 Multiphor Il application kits and accessories Optional accessories Designation Code No Roller 80 1106 79 GelBond PAGfilm 124 x 258 mm 50 pkg 80 1129 36 Bind Silane 100 ml 17 1330 01 Repel Silane 500 ml 17 1331 01 SDS Sample Application Strip 26 samples 40 ul 18 1
58. the stiff plastic film facing down in the middle of the cooling plate making sure no air bubbles are trapped under the gel Position the gel so that the polarity of the gel corresponds to that of the plate Use the screened template on the cooling plate to centre the gel Remove any excess solution with paper tissue Remove the protective cover film from the gel Open the ExcelGel SDS strip packages and apply the cathodic and the anodic SDS buffer strips on the respective sides of the gel xy Y atthe Note The narrowest side of the buffer strip should be placed on the gel surface Choose an appropriate sample application method and apply the sample Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 21 5 Operation 22 Align the EPH IEF electrodes with the centre of the buffer strips by loosening the clamping nuts and sliding the electrodes to the appropriate position Retighten the clamping nuts Lift the electrode holder slightly and reposition the supporting feet over the deep holes Lower carefully so that the electrodes rest on the buffer strips Connect the electrodes to the buffer tank During electrophoresis the socket on the bridging cable MUST be attached to the pin connector at the front of the buffer tank as shown in the picture Connect the two electrodes to the buffer tank using the spring loaded cables on the electrodes Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition
59. thioether DPT papers Transferring proteins from SDS polyacrylamide gels to nitrocellulose membrane The support or backing film must be removed from all polyacrylamide and agarose gels before electrophoretic transfer Using FilmRemover the film is removed quickly and cleanly Instructions for use are supplied with FilmRemover Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 1 Saturate the graphite anode plate with distilled water and remove the excess water with absorbent paper With the electrode lead to the front of the instrument fit the lower anode plate onto the buffer tank Connect the anode socket red lead to the anode pin on the right side of the buffer tank The transfer sandwich can now be assembled on the anode electrode Note When assembling the transfer sandwich in NovaBlot unit always wear gloves 2 To ensure that the current passes only through the gel cut the filter papers and the immobilizing and dialysis membranes to the same size as the gel to be transferred When using a discontinuous buffer system carefully soak the first layer of six filter papers in anode solution R see Section 4 9 Stock solutions by slowly immersing the papers under the surface of the electrode solution Allow them to become wet by capillary action and avoid trapping air bubbles that may interfere with the transfer Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 37
60. trophoresis Unit to the cooling device install tubing connectors on both inlet and outlet tubings Use one male and one female connector on the Multiphor Il unit side and thermostat unit side respectively The cooling plate tubings and thermostatic circulator tubings can then be locked separately Two Multiphor Il units can also be connected in series to one thermostatic circulator Place the rubber grommets on the cooling plate inlet and outlet tubes Slide a short piece of tubing onto each tube and secure with hose clamps Attach the male and female tubing connectors as described above Repeat this process with the thermostatic circulator using longer pieces of tubing To lock the connectors insert the male connector into the female and turn clockwise one quarter turn until it clicks Screw one levelling foot into each corner of the buffer tank Place the buffer tank on the lab bench where it will be used Place the cooling plate on the unit using the moulded guides to position it correctly Fit the grommets into the cutouts in the back of the unit Place a spirit level on the cooling plate and adjust the levelling feet until the unit is levelled Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 15 4 Installation To mount the EPH IEF electrodes on the electrode holder unscrew the clamping nut from each electrode When running electrophoresis across the width of the cooling plate mount the electrodes as ill
61. uate measures Recycling This symbol indicates that the waste of electrical amp and electronic equipment must not be disposed as unsorted municipal waste and must be EE collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment Contents Contents 1 Introduction 2 Safety information 3 Description of parts 4 Installation 5 Operation 5 1 Electrophoresis using ExcelGel SDS and buffer Strips 20 5 2 Electrophoresis using buffer CNAMDETS ou esssesseesseesseessesseessesseeseeeseen 25 5 3 Isoelectric focusing using Ampholine PAGPIAtEe 28 5 4 Isoelectric focusing using CleanGel IEF ooccocccocaconaconicaninnnnnnanoninnonononenoses 32 5 5 2 D electrophoresis using Immobiline DryStrip and ExcelGel SDS 34 5 6 Electrophoretic transfer Introduction iii i 35 ia RE ene Ee nen anne Pee rene 42 5 8 Running conditions for precast GElS iii 43 6 Maintenance CI PR SU o cc ncn ee 47 7 Technical specifications 8 Trouble shooting 9 Multiphor Il application kits and accessories 92 Buffer Strip POSITION 59 9 3 Immobiline DruStrip Reswelling TlU cccnnnnmmmsma 60 94 oneri 60 95 NovdBIOt KIU acca cece sc encpearese enerne NoN apearen sevens er ssettsaresreeseneenariestnsee 61 A ia 62 A RR 63 98 Sample application ACCESS OF CS iii 63 10 Ordering infor
62. uring electrophoresis a uniform layer of a non charged insulating fluid is applied under the gel To do this pour a few milliliters of kerosene or light paraffin oil towards one end of the cooling plate Starting with one end of the gel support gradually lower the gel to the horizontal position constantly checking for trapped air bubbles If air becomes trapped raise the gel just enough to release the air and then continue to lower it onto the cooling plate Use the template markings to centre the gel on the cooling plate Remove any excess solution with a tissue Repeat this procedure for each gel If voltage probe measurements are required the gel s must be positioned with the direction of the current path across the width of the cooling plate Prepare the electrode wicks by aligning 8 10 pieces of filter paper for each buffer chamber Starting at one end slowly immerse the electrode wicks in the buffer using capillary action to reduce the amount of air trapped in the paper wr n y y When running large 195 x 250 mm or medium sized gels 120 x 250 mm place the wicks so that the long edge overlaps the gel by 15 mm over the entire length The template markings are useful for checking that the alignment is correct For running small gels 84x94 mm on the large cooling plate place the wicks with the short edge overlapping the gel by 15 mm In this case one set of wicks is required for each gel Apply the samples a
63. ustrated Place the electrode under the electrode holder Replace the nut and lightly tighten until the electrode is held firmly in place To align the electrodes with the electrode strips place the electrode holder with the electrodes onto the false holes on the buffer tank using the four corner feet When running electrophoresis along the length of the cooling plate mount the electrodes as illustrated Place the electrodes under the electrode holder 16 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Installation 4 Replace the nut and lightly tighten until the electrode is held firmly in place To align the electrodes with the electrode strips place the electrode holder with the electrodes onto the false holes on the buffer tank using the four corner feet Install the safety lid Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 17 4 Installation 18 Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 5 Operation such as wearing protective glasses and gloves resistant to the chemicals used Follow Y WARNING When using hazardous chemicals take all suitable protective measures local regulations and instructions for safe operation and maintenance of the system This section together with the information supplied with the precast gels gives all the necessary information to run most analytical electrophoresis techniques using our pre
64. ve condensation Incorrect electrode solutions Excessive power setting Excess moisture on gel or under cooling plate Electrode strips overhanging the ends of the gel Liquid expelled at sides of electrode strips due to electroendosmotic of water towards the cathode Excessive power setting Insufficient cooling Local overheating due to a high salt concentration in the sample Incorrect electrode solutions in relation to electrode polarity Localized hot spots due to air bubbles under the gel Cathodic drift may cause an electroendosmotic flow of water towards the cathode Thus cathode strip may become over saturated Reversed polarity of electrode strips lower pH at cathode higher pH at anode Remedy Check the temperature and flow of the cooling fluid Lower the power Decrease condensation by adjusting the temperature of the cooling fluid Wipe the electrode holder periodically to remove condensation Use the recommended electrode solution at the specified concentration Check the power setting Remove the excess moisture Cut the electrode strips short of the ends of the gel Occasionally remove the excess flow fluid by blotting Check the power setting When only a portion of the gel is used reduce the power setting proportionally Check the temperature and flow of the cooling fluid Reduce the salt content of the sample by gel filtration using PD 10 columns pre packed with Sephadex G 25
65. with high electrical resistance in the cooling plate WARNING NEVER EXCEED the maximum pressure 0 5 bar in the cooling plate WARNING NEVER EXCEED the maximum allowed voltage current or power WARNING The cooling plate is rated for operation at up to 3 5 kV p p gt gt gt 2 pee ReRe BB Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 9 2 Safety information WARNING When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the chemicals used Follow local regulations and instructions for safe operation and maintenance of the system been flushed thoroughly with bacteriostatic solution e g NaOH and distilled water before service and maintenance 1 WARNING When using hazardous chemicals make sure that the entire system has 10 Multiphor Il Electrophoresis System User Manual 18 1103 43 Edition AK 3 Description of parts Description of parts 3 The Multiphor Il Electrophoresis Unit includes buffer tank with 4 levelling feet cooling plate with accessories safety lid electrode holder with movable EPH IEF electrodes for buffer strips and electrode strips Unit contents Code No 18 1018 06 Designation Buffer Tank Levelling Foot 4 pkg Cooling Plate ceramic 210 x 270 mm Grommet 2 pkg Cooling Tubing 8 12 mm 4 m Tubing Connector Set 2 pcs female 2 pcs male Hose Clamp 1
66. x 2H 0 Make up to 250 ml with distilled water H Preserving solution Glycerol 87 w w Make up to 250 ml with distilled water Destaining solution Ethanol Acetic acid Make up to 1000 ml with distilled water K Coomassie solution PhastGel Blue R Make up to 400 ml with destaining solution Heat to 60 C stirring constantly and filter before use L Preserving solution Glycerol 87 w w Make up to 250 ml with destaining solution N Fixing solution Trichloroacetic acid Make up to 500 ml with distilled water 400 ml 100 ml 75 ml 17 00 g 125ml 0 50 g 0 25 g 50 ul 6 25g Zul 3 65 9 25 ml 250 ml 80 ml 1 tablet 25 ml 100 g Multiphor ll Electrophoresis System User Manual 18 1103 43 Edition AK Operation 5 Transfer buffers using a discontinuous buffer system R Anode solution 1 pH 10 4 Tris 50 0 0 Methanol 200 ml Make up to 1000 ml with distilled water S Anode solution 2 pH 10 4 Tris 5 030 Methanol 200 ml Make up to 1000 ml with distilled water T Cathode solution pH 7 6 6 Amino n hexanoic acid 5 20 g Methanol 200 ml Make up to 1000 ml with distilled water U Transfer buffer using a continuous buffer system Glycine 2 939 Tris 5810 SDS 0 3759 Methanol 200 ml Make up to 1000 ml with distilled water Note In a continuous buffer system this solution is used for both anode and cathode electrode solutions 5 8 Running conditions for precast gels ExcelGel

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