Pharmacia Biotech 18-1016-85 Скачать руководство пользователя страница 9

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1. Introduction

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Содержание 18-1016-85

Страница 1: ...nd idle equipment along with credit for buybacks and trade ins Custom engineering so your equipment works exactly as you specify Critical and expedited services Leasing Rentals Demos In stock Ready to...

Страница 2: ...P H A R M A C I A B I O T E C H Multiphor II Electrophoresis System User Manual 18 1103 43 Edition AF A Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 3: ...Inc Mylar is a trademark of E I du Pont de Nemours Co Copyright 1996 Pharmacia Biotech AB All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted...

Страница 4: ...35 5 6 Isoelectric focusing using Immobiline DryPlate 37 5 7 2 D Electrophoresis using Immobiline DryStrip and ExcelGel SDS gradient 38 5 8 Electrophoretic transfer 40 5 9 Stock solutions 47 5 10 Runn...

Страница 5: ...phor II 95 10 2 MultiTemp III 99 10 3 EPS Power Supplies 99 10 4 Hoefer Automated Gel Stainer 99 10 5 Precast gels and buffer strips 100 10 6 Molecular weight and pl markers 100 10 7 Carrier ampholyte...

Страница 6: ...radient ExcelGel SDS Homogeneous CleanGel IEF Ampholine PAGplate Immobiline DryPlate CleanGel IEF 2 D electrophoresis Immobiline DryStrip ExcelGel XL SDS gradient Multiphor II is available in two basi...

Страница 7: ...ip Kit 18 1004 30 DryStrip Reswelling Cassette 18 1013 74 2 D second dimension SDS and Native PAGE IEF Kit 18 1102 45 0 5x125x260 mm Gradient Maker 18 1013 72 2 D second dimension Large Scale SDS and...

Страница 8: ...bly and use of each Multiphor II application kit 8 Ordering information Multiphor II basic configurations application kits accessories and replacement parts MultiTemp III EPS and GPS Power Supplies Pr...

Страница 9: ...6 1 Introduction Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 10: ...les 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 hydr...

Страница 11: ...8 2 Safety information WARNING The cooling plate is rated for operation at up to 3 5 kV p p Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 12: ...80 1106 58 Cooling Tubing 8 12 mm 4 m 80 1106 56 Tubing Connector Set 2 pcs female 2 pcs male 18 1104 26 Hose Clamp 10 pkg 18 1104 27 Safety Lid 18 1122 26 Electrode Holder 80 1106 55 EPH IEF Electro...

Страница 13: ...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 recesse...

Страница 14: ...iculator such as MultiTemp III The cooling tubing tubing connector set and hose clamps provide a flexible and safe way to connect Multiphor II to a thermostatic circulator such as MultiTemp III The el...

Страница 15: ...for safety 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 tan...

Страница 16: ...IEF electrodes and EPH electrodes the NovaBlot unit can be expanded into a horizontal electrophoresis system see 2 1 Multiphor II Electrophoresis System Unit contents Code No 18 1016 85 Designation C...

Страница 17: ...lene which is resistant to nearly all chemicals at room temperature The safety lid made of polycarbonate includes the electrode leads and the safety interlock Precut electrode paper 200x250 mm and cel...

Страница 18: ...er 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...

Страница 19: ...he 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 buff...

Страница 20: ...is rinsed with water and dried Saturate the anode NovaBlot electrode with distilled water and remove the excess water with absorbent paper With the electrode lead to the front of the instrument fit t...

Страница 21: ...rbent paper With the electrode lead to the front of the instrument place the cathode electrode on top of the stack Connect the cathode socket black lead to the cathode pin on the left of the buffer ta...

Страница 22: ...buffer strips ExcelGel buffer strips made of polyacrylamide and CleanGel buffer strips made of special paper The buffer strips are applied on the gel edges with the electrodes on top Immunoelectropho...

Страница 23: ...ue 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...

Страница 24: ...p II 15 minutes before starting the experiment and set the temperature to 15 C Always wear clean gloves when working with polyacrylamide gels and buffer strips particularly when using sensitive staini...

Страница 25: ...tly 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...

Страница 26: ...plied with the precast gel Running conditions Voltage Current Power Time V mA W min Run 600 50 30 75 Approximate time or until the Bromophenol Blue front reaches the anode buffer strip When the Bromop...

Страница 27: ...n I 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...

Страница 28: ...ion of the running procedure for native PAGE and SDS PAGE using CleanGel and CleanGel buffer strips CleanGel is a washed and dried polyacrylamide gel Depending on the application the gels are rehydrat...

Страница 29: ...compartments of the PaperPool If a smaller gel size is used cut the strips to the correct size Apply 20 ml of the cathode buffer to the cathodic buffer strip using a pipette use less buffer volume for...

Страница 30: ...ecommended electrical settings for anodal electrophoresis with Native Buffer Kit pH 8 9 Phase Voltage Current Power Time V mA W min Pre run 300 18 10 10 Run 900 50 30 60 Approximate time or until the...

Страница 31: ...icks 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...

Страница 32: ...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...

Страница 33: ...used as a tracking dye 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 1...

Страница 34: ...5 9 5 1 M Phosphoric acid 1 0 M NaOH 4 0 6 5 0 1 M Glutamic acid 0 1 M 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 Wea...

Страница 35: ...ecially 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 appli...

Страница 36: ...s 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 is...

Страница 37: ...me h 3 5 9 5 1 500 50 30 1 5 4 0 6 5 2 000 25 25 2 5 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...

Страница 38: ...n I 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...

Страница 39: ...mended 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 25...

Страница 40: ...at this stage Instructions are supplied with Immobiline DryPlate gels Immobiline DryPlate is run with electrode strips wetted in distilled water Recommended running conditions for one Immobiline DryP...

Страница 41: ...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 II The high sam...

Страница 42: ...of a stacking gel zone followed by the polyacrylamide gradient ExcelGel SDS gels Gel Gradient Size mm ExcelGel SDS 8 18 245 x 110 ExcelGel XL SDS 12 14 245 x 180 ExcelGel SDS buffer strips supply the...

Страница 43: ...ectrophoretic transfer using NovaBlot system is dependant on Characteristics of the immobilizing membrane Characteristics of the transfer buffer Molecular weight and charge of the protein Gel thicknes...

Страница 44: ...most commonly used however low molecular proteins may be lost By using pore sizes of 0 2 or 0 1 m most proteins are retained Nitrocellulose membranes can be probed several times The membranes require...

Страница 45: ...lot 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 Whe...

Страница 46: ...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 ass...

Страница 47: ...of the gel to complete the transfer sandwich 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...

Страница 48: ...pply It is recommended to run the transfer at a constant current of 0 8 mA cm2 A transfer time of approximately 1 hour is normal Note The current is calculated using the surface area total length x wi...

Страница 49: ...r 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...

Страница 50: ...to 250 ml with distilled water F Developing solution Sodium carbonate 6 25 g Formaldehyde 25 l Make up to 250 ml with distilled water G Stop solution EDTA Na2 x 2H2O 3 65 g Make up to 250 ml with dist...

Страница 51: ...nd cathode electrode solutions ExcelGel SDS gradient 8 18 Running conditions Phase Voltage Current Power Time Temp V mA W min C Run 600 50 30 75 15 Or until the Bromophenol Blue front reaches the anod...

Страница 52: ...esis with Native Buffer Kit pH 5 5 Phase Voltage Current Power Time Temp V mA W min C pre run 500 10 10 10 10 run 1200 28 28 50 10 Or until the Pyronin front reaches the cathodic wick CleanGel with Na...

Страница 53: ...3500 1 5 20 10 2 D electrophoresis using Immobiline DryStrip and ExcelGel SDS gradient First dimension Option 1 EPS 3500 XL Power Supply using a voltage gradient The parameters below may be used for...

Страница 54: ...10 35000 Total 16 45500 The optimal total number of Volt hours for these pH gradients depends on the type of sample sample load g and sample volume Option 2 Using a Manual Power Supply The power suppl...

Страница 55: ...30 25 30 15 2 600 50 30 3 5 15 3 600 50 30 70 15 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 DrySt...

Страница 56: ...5 Operation 53 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 57: ...5 Operation 54 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 58: ...5 Operation 55 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 59: ...e 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 pap...

Страница 60: ...6 Maintenance 54 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 61: ...acids alkalis and alcohols Safety standards This products meets the requirements of the Low Voltage Directive LVD 73 23 EEC through the harmonized standards EN61010 1 Note The declaration of conformit...

Страница 62: ...7 Technical specifications 56 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 63: ...nd the wicks and electrodes Poor cooling Check the cooling Burning at slots or Polypeptide complexes are too big to If SDS PAGE add DTT once accumulation of enter the gel and cause again and boil the...

Страница 64: ...he sample sample by gel filtration using PD 10 columns pre packed with Sephadex G 25 Incorrect electrode solutions in relation Check the electrode polarity to electrode polarity Check the pH of the ap...

Страница 65: ...time too short Increase transfer time Field strength too low Increase field strength Too low charge mass ratio Change transfer buffer pH further away from molecules pI Add 0 1 SDS Poor transfer Air t...

Страница 66: ...8 Trouble shooting 60 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 67: ...8 Trouble shooting 61 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 68: ...ss plate 125x260 mm Alternatively casting can be done on GelBond PAGfilm 124x258 mm Optional glass plates with U frames allow casting of 1 0 and 2 0 mm thick gels The kit includes sample application p...

Страница 69: ...he 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...

Страница 70: ...k 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 i...

Страница 71: ...1 mm 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 introduc...

Страница 72: ...Plate 200x260x4 mm 2 pkg 18 1102 47 FlexiClamp 6 pkg 18 1013 73 Electrophoresis Wick 104x253 mm 500 pkg 80 1129 52 IEF SDS Sample Application Strip 52 samples 5 20 l 5 pkg 18 1002 26 Optional accesso...

Страница 73: ...IEF with the carrier ampholytes Ampholine or Pharmalyte The sample is applied by soaking IEF Sample Application Pieces in 15 20 l of sample solution and placing them on the gel surface Alternatively s...

Страница 74: ...thick glass plate and lay the film over it with the hydrophilic side up see Instructions supplied with the film Centre the film over the glass plate Beginning at one end use the roller to apply an eve...

Страница 75: ...solution to gel for 15 minutes at room temperature Before opening remove the excess gel from the ends of the mould with a scalpel Remove the two FlexiClamps Stand the mould on one long side Grasp the...

Страница 76: ...Note GelBond film is recommended for all one dimensional immunoelectrophoretic techniques and as the second dimension in crossed immunoelectrophoresis However for ease of handling the gel in two dime...

Страница 77: ...coat the glass plates with agarose so that the gels adhere during staining and destaining Apply a thin layer of hot agarose solution 0 l 0 3 onto each warmed glass plate distribute it evenly and leav...

Страница 78: ...to the vacuum tubing from a vacuum source Select the appropriate Template and place it in the Plate Holder One of the corners of the Plate Holder is rounded to ensure that the Template is in the corr...

Страница 79: ...on Diffusion techniques can be performed in the Humidity Chamber 7 For Grabar Williams immunoelectrophoresis troughs must be formed in the gel after the first dimension electrophoresis Using the Templ...

Страница 80: ...ode No Tray 125x260 mm rim 5 mm 2 pkg 80 1107 03 IEF Electrode Strip 100 pkg 18 1004 40 Sample Applicator 80 1107 15 PEGG Print Paper Electrophoresis Wick 104x253 mm 500 pkg 80 1129 52 Fractionating G...

Страница 81: ...the suspension with a gentle stream of air The speed and distance of the fan should be adjusted so that no ripples are formed on the surface of the suspension Calculate the final weight of the prepar...

Страница 82: ...he applicator and allow the bed to equilibrate hydrostatically for a few minutes Place the electrode holder with the EPH IEF electrode onto the Electrode Strips and start the isoelectric focusing When...

Страница 83: ...er directly along the tray Press the fractionating grid frame into the gel bed The frame is slightly shorter than the tray which makes it possible to match the slots for the blades with the protein zo...

Страница 84: ...under a protective layer of silicone oil The high sample capacity allows the application of up to 100 l on each Immobiline DryStrip Detailed instructions for use are available in the instruction manu...

Страница 85: ...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 cat...

Страница 86: ...mm 80 1115 81 Holding Guides 4 pcs 80 1318 35 Weight 80 1115 88 Gel Spacer Set 5 units pkg 56 1154 98 Optional accessories Designation Code No Roller 80 1106 79 GelBond PAGfilm 124x258 mm 50 pkg 80 11...

Страница 87: ...pressure over the surface in order to eliminate air bubbles and seal the film to the plate with a minimum amount of water Remove any excess water with a tissue Place the gel spacer of the desired thi...

Страница 88: ...m contact with the toxic acrylamide solution To obtain the correct gel thickness apply an even and continuous pressure to the short glass plate during the entire casting procedure With one hand presse...

Страница 89: ...en the gel and the short glass plate and allows the plate to be removed without disturbing the gel surface Holding the gel spacer at one corner carefully peel it away Remove the prepared gel from the...

Страница 90: ...ating Stick 80 1108 67 Silicone Tubing 1 35 3 35 mm 2 m 80 1065 91 Pinch Cock 2 pkg 80 1106 41 Polyethylene Tubing 1 2 mm 5 m 80 1065 75 Cut a 5 cm length of the silicone tubing attach the pinch cock...

Страница 91: ...levelled Place the prepared mould on a levelled table and insert the end of the tubing between the glass plates in the centred groove The tubing should lie against the gel support glass plate or film...

Страница 92: ...water Also check the outlet and tubing for small pieces of gel Dry the components with a tissue and reassemble The Reswelling Cassette is used for reproducible rehydration of precast Immobiline DryPla...

Страница 93: ...lush with the inner face of the plate Apply the pinch cock to the silicone tubing If required the tubing can be permanently fixed using a silicone glue such as General Electric RTV Silicone Rubber Adh...

Страница 94: ...d glass plate on top of the dry gel and glass plate Clamp the cassette together using five FlexiClamps Stand the assembled Reswelling Cassette vertically Fill the syringe with approximately 20 ml of t...

Страница 95: ...ls agarose gels and when preparing granulated gel beds for preparative IEF Unit contents Code No 18 1016 88 Designation Code No Levelling Table 80 1316 58 Spirit Level 56 2019 48 The Humidity Chamber...

Страница 96: ...e in the instruction manual provided with the product Unit contents 18 1013 75 Designation Code No FilmRemover basic unit 80 1316 21 Lever and Wire Assembly 3 pkg 18 1013 79 Instruction Manual 80 1316...

Страница 97: ...18 1018 08 Staining Tray 2 60x260x320 mm 18 1018 09 For use when applying plastic support films onto glass plates with an interfacing fluid The roller is used to provide even pressure over a large ar...

Страница 98: ...application Designation Code No IEF Sample Application Pieces 200 pkg 80 1129 46 The 5x10 mm sample application piece made of Paratex can be used for sample volumes in the range 15 20 l Designation Co...

Страница 99: ...ples can be applied and each well holds up to 40 l of sample The strip is made of transparent flexible silicone and is applied directly onto the gel surface Designation Code No IEF SDS Sample Applicat...

Страница 100: ...a sample volume of 2 4 l 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...

Страница 101: ...9 Multiphor II application kits and accessories 94 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 102: ...mm NovaBlot Kit 1 18 1016 86 for electrophoretic transfer Accessories and replacement parts UltroMould 1 18 1018 16 for casting 124x258 mm ultrathin 0 1 0 2 0 3 0 4 and 0 5 mm polyacrylamide gels Grad...

Страница 103: ...Spacer UltroMould 0 2 mm 4 80 1115 23 Gel Spacer UltroMould 0 3 mm 3 80 1115 24 Gel Spacer UltroMould 0 4 mm 2 80 1115 25 Gel Spacer UltroMould 0 5 mm 1 80 1115 26 Lever and Wire Assemblies for Film R...

Страница 104: ...anode 1 80 1257 87 NovaBlot Electrode cathode 1 18 1019 86 Glass plates and trays 84x94x1 mm 50 80 1106 69 125x260x3 mm 2 80 1106 99 125x260x1 mm 15 80 1106 29 200x260x4 mm 2 18 1102 47 125x260 mm 0 5...

Страница 105: ...used as PEGG print paper and with agarose IEF 500 80 1129 52 Electrode Paper NovaBlot 200x250 mm 500 80 1106 19 Sample application Syringe 15 l 1 80 1106 48 SDS Sample Application Strip 26 samples 40...

Страница 106: ...mostatic circulator 220 220 V Hose Clamps 10 18 1104 27 Cooling Tubing 8 12 mm 4 m 80 1106 56 Tubing Connector Set female and male 4 18 1104 26 Insulation for Cooling Tubing 14 27 mm 2 m 80 1116 11 3...

Страница 107: ...Pool for electrode strips 1 18 1031 59 IEF CleanGel IEF 5 18 1035 32 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 Am...

Страница 108: ...l 80 1127 17 Ampholine preblended pH 5 0 8 0 25 ml 80 1127 19 Ampholine for PGM 25 ml 17 0997 01 Ampholine pH 3 5 10 0 25 ml 80 1125 87 Ampholine pH 3 5 5 0 25 ml 80 1125 89 Ampholine pH 4 0 6 0 25 ml...

Страница 109: ...urell rockets Crossed immunoelectrophoresis n n Clarke Freeman Immunodiffusion o o n o o Ouchterlony Counter immunoelectrophoresis n Nucleic acid o o n n electrophoresis Cell culture and n n cloning t...

Страница 110: ...rbitone buffer Serum electrophoresis 250 ml A 17 1334 01 Additives and sample treatment Urea IEF PAGE Sequencing 500 g B 17 1319 01 Formamide IEF PAGE Sequencing 250 ml B 17 1320 01 Dithiothreitol IEF...

Страница 111: ...10 Ordering information 104 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 112: ...10 Ordering information 105 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 113: ...A Printed in Sweden by TK i Uppsala AB February 1997 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com...

Страница 114: ...uipment Have surplus equipment taking up shelf space We ll give it a new home Learn more Visit us at artisantg com for more info on price quotes drivers technical specifications manuals and documentat...

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