Pharmacia Biotech Multiphor II User manual

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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
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Importantuserinformation
Warranty and Liability
Pharmacia Biotech AB guarantees that the product
delivered has been thoroughly tested to ensure that it
meets its published specifications. The warranty included
in the conditions of delivery is valid only if the product
has been installed and used according to the instructions
supplied by Pharmacia Biotech AB.
Pharmacia Biotech AB shall in no event be liable for
incidental or consequential damages, including without
limitation, lost profits, loss of income, loss of business
opportunities, loss of use and other related exposures,
however caused, arising from the faulty and incorrect use
of the product.
Trademarks
The following designations are trademarks owned by
Pharmacia Biotech. Multiphor, NovaBlot, Immobiline,
DryStrip,FlexiClamp,UltraMould,DryPlate,Ampholine,
MultiTemp,ExcelGel, PAGplate,Pharmalyte,Bind-Silane,
Repel-Silane, FilmRemover, PlusOne, ReadyMix,
ReadySol.
CleanGel,PaperPool andGelPoolaretrademarksof ETC
Gmbh.GelBond PAGfilmisa trademarkof FMCCorp.
Triton,TrisandAmberlitearetrademarksofRohm&Haas.
TweenisatrademarkofAtlasChemicalIndustriesInc.Mylar
isa trademarkofE.I. duPont deNemours& Co.
Copyright©1996
Pharmacia BiotechAB
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system or transmitted in
any form by any means, without permission in written
form from Pharmacia Biotech AB.
Pharmacia Biotech AB reserves the right to make changes
to the design and specifications without prior notice.
Meaning: Consult the insruction manual to avoid
persoanl injury, or damage to the product or other
equipment.
WARNING! The Warning sign is used to call attention
to the necessity to follow an instruction in detail to
avoid personal injury. Do not proceed until the
instructions are clerarly understood and all stated
conditions are met.
CAUTION! The Caution sign is used to call attention
to instructions or conditions that are to be followed to
avoid damage to the product or other equipment. Do
not proceed until the instructions are clerarly
understood and all stated conditions are met.
Note: The Note sign is used to indicate information
important for trouble free or optiamal use of the
prodct. Should you have any comments on this
instruction, we will be pleased to receive them at:
Pharmacia Biotech AB
S-751 82 Uppsala
Sweden
w
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Contents
1. Introduction ..............................................................................3
2. Safety information.....................................................................7
3. Description of parts...................................................................9
3.1 Multiphor II Electrophoresis Unit..................................9
3.2 Multiphor II NovaBlot Unit for
electrophoretic transfer ...............................................13
4. Installation ..............................................................................15
4.1 Multiphor II Electrophoresis Unit................................15
4.2 Multiphor II NovaBlot Unit for
electrophoretic transfer................................................17
5. Operation................................................................................19
5.1 Electrophoresis using ExcelGel SDS
and buffer strips..........................................................20
5.2 Electrophoresis using CleanGel and buffer strips.........25
5.3 Electrophoresis using buffer chambers.........................28
5.4 Isoelectric focusing using Ampholine PAGplate...........31
5.5 Isoelectric focusing using CleanGel IEF.......................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 Running conditions for precast gels...........................48
6. Maintenance............................................................................53
7. Technical specification.............................................................55
8. Trouble shooting.....................................................................57
9. Multiphor II application kits and accessories...........................61
9.1 SDS and Native PAGE, IEF Kit....................................61
9.2 Large Scale SDS and Native PAGE Kit ........................65
9.3 Agarose IEF Kit...........................................................66
9.4 Immunoelectrophoresis Kit..........................................69
9.5 Preparative IEF Kit......................................................73
9.6 Immobiline DryStrip Kit..............................................77
9.7 NovaBlot Kit...............................................................78
9.8 UltroMould.................................................................79
9.9 Gradient Maker...........................................................83
9.10 Reswelling Cassette...................................................85
9.11 Levelling Set..............................................................88
9.12 Humidity Chamber ...................................................88
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9.13 FilmRemover.............................................................89
9.14 Staining Tray 1 and 2................................................90
9.15 Roller........................................................................90
9.16 Sample application accessories ..................................91
9.17 Misellaneous .............................................................93
10. Ordering information............................................................95
10.1 Multiphor 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 ampholytes..................................................100
10.8 Agarose, Ultrodex and PlusOne
electrophoresis chemicals...........................................101
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3
1. Introduction
Multiphor II 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, a unique range of precast gels for
all major electrophoretic techniques is available with Multiphor II:
Technique Precast Gel
SDS and Native PAGE ExcelGel SDS Gradient
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 basic configurations: Multiphor II
Electrophoresis Unit for electrophoresis and isoelectric focusing, and
Multiphor II NovaBlot Unit for electrophoretic transfer.
If laboratory cast gels are preferred, application kits and accessories can be
added to either of the basic configurations.
1. Introduction
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The following guide summarizes how you can expand and use Multiphor II
Electrophoresis Unit with application kits and accessories.
Application Recommended Kit/Accessory Code No.
SDS and Native PAGE SDS and Native PAGE, IEF Kit 18-1102-45
0.5x125x260 mm Gradient Maker 18-1013-72
homogeneous and gradient gel
SDS and Native PAGE Large Scale SDS and Native 18-1102-46
0.5x200x260 mm PAGE Kit
homogeneous and gradient gel Gradient Maker 18-1013-72
IEF in polyacrylamide UltroMould 18-1018-16
0.1-0.5x125x260 mm
IEF in polyacrylamide SDS and Native PAGE, IEF Kit 18-1102-45
0.5x125x260 mm
IEF in agarose Agarose IEF Kit 18-1016-82
Preparative IEF in granulated Preparative IEF Kit 18-1018-05
gel 5x125x260 mm
Immunoelectrophoresis - Immunoelectrophoresis Kit 18-1016-87
Grabar & Williams, Laurell
and fused rocket, crossed and
tandem crossed, double
diffusion
2-D, first dimension: Immobiline Immobiline DryStrip 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 Native 18-1102-46
0.5x200x260 mm PAGE Kit
Gradient Maker 18-1013-72
Electrophoretic transfer NovaBlot Kit 18-1016-86
FilmRemover 18-1013-75
This User Manual is comprised of the following sections:
1. ‘‘Introduction’’ includes a general description of Multiphor II System and
dedicated precast gels, a guide to the application kits and the manual
structure.
2. ‘‘Description of parts’’ describes in detail the components of Multiphor II
Electrophoresis Unit and Multiphor II NovaBlot Unit.
3. ‘‘Installation’’ contains a detailed description of how to install Multiphor
II Electrophoresis Unit and Multiphor II NovaBlot Unit.
4. ‘‘Operation’’ contains information on the operating procedure for SDS
and native polyacrylamide gel electrophoresis, isoelectric focusing, 2-D
electrophoresis and electrophoretic transfer.
1. Introduction
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5
5. ‘‘Maintenance’’ gives cleaning recommendations to help you maintain
your Multiphor II unit.
6. ‘‘Trouble shooting’’ offers suggestions for correcting problems that may
occur.
7. ‘‘Multiphor II application kits and accessories’’ describes in detail the
contents, assembly 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
- Precast gels and buffer strips
- Molecular weight and pI markers
- Ampholine and Pharmalyte carrier ampholytes
- Gel casting and electrophoresis chemicals
1. Introduction
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6
1. Introduction
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7
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 only.
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 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! 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.
2. Safety information
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8
2. Safety information
WARNING! The cooling plate is rated for operation at up to 3.5
kV (p-p).
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3. Description of parts
9
3. Description of parts
This Multiphor II configuration 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) and card-mounted
EPH electrodes (for electrophoresis using buffer chambers).
Unit contents - Code No. 18-1018-06
Designation Code No
Buffer Tank 18-1122-25
Levelling Foot (4/pkg) 18-1026-40
Cooling Plate ceramic, 210x270 mm 18-1103-46
Grommet (2/pkg) 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 Electrode cathode 80-1121-52
EPH/IEF Electrode anode 80-1121-53
User Manual and Application Package 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.
3.1 Multiphor II
Electrophoresis
Unit
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3. Description of parts
10
The larger pins are for connection to the safety lid and complete the
electrical circuit when the lid is in position.
The small left land 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 (red) 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 CO2content 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.
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3. Description of parts
11
The ceramic (aluminium oxide) cooling plate measures 210x270
mm, supports the gel, and provides uniform temperature control.
Aluminium oxide is an excellent heat conductor and electrical
insulating material.
To facilitate the correct positioning of electrophoresis gels, the
surface of the cooling plate is screened with a template measuring
190x250 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.
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 electrode holder 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.
WARNING! The cooling plate is rated for operation at up to 3.5
kV (p-p).
WARNING! Only use water with high electrical resistance as
coolant and NEVER EXCEED maximum pressure of 0.5 bar.
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3. Description of parts
12
The electrode holder consists of a double strength glass plate with ground
edges and four corner feet made of Rynite 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 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 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.
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3. Description of parts
13
This Multiphor II configuration is a dedicated unit for electrophoretic
transfer and includes a buffer tank with four levelling feet, a safety lid,
NovaBlot electrodes, electrode paper and cellophane sheets.
The unique NovaBlot electrodes made of graphite (210 x 260 mm), provide
an even distribution of current across the entire surface, thus ensuring
uniform electrophoretic transfer.
Both electrodes are bound to acrylic plates. The lower anode electrode forms
the base and the upper cathode electrode forms the lid of the transfer
assembly.
The NovaBlot electrodes are designed so that when cathode and anode
electrodes are fitted together, they will not touch each other. This prevents
damage to the graphite electrodes when they are not in use.
By addition of the cooling plate, electrode holder with EPH/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 Code No.
Buffer Tank 18-1122-25
Levelling Feet (4/pkg) 18-1026-40
Safety Lid 18-1122-26
NovaBlot Electrode, cathode 18-1019-86
NovaBlot Electrode, anode 80-1257-87
Electrode Paper NovaBlot, 200x250 mm (500/pkg) 80-1106-19
Cellophane Sheets, 210x320 mm (50/pkg) 80-1129-38
Manual and Application Package 18-1103-44
Optional accessories
Designation Code No.
FilmRemover 18-1013-75
Nitrocellulose 0.20 µm, 150x200 mm (15/pkg) 80-1098-91
Nitrocellulose 0.45 µm, 150x200 mm (15/pkg) 80-1098-90
ProBind 45 NC 0.45 µm, roll 0.2x3.0 m 80-1247-86
GeneBind 45 nylon 0.45 µm, roll 0.2x3.0 m 80-1247-87
The Multiphor II buffer tank with the four adjustable levelling feet supports
the NovaBlot electrodes.
3.2 Multiphor II
NovaBlot Unit
for electro-
phoretic
transfer
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3. Description of parts
14
Two electrode connections are provided for each pole, one small pin and one
large banana plug. The larger banana plugs connect the buffer tank to the
safety lid. The NovaBlot electrodes are connected to the small pins.
The buffer tank is made of polypropylene, 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 cellophane sheets, 210x320 mm,
are included with Multiphor II NovaBlot Unit.
WARNING! Polycarbonate is not resistant to concentrated acids
and bases, or to halogenated and aromatic hydrocarbons.
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4. Installation
15
4.1 Multiphor II
Electro-
phoresis Unit
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 II Electrophoresis Unit to the cooling
device, install tubing connectors on both inlet and outlet tubings. Use one
male and one female connector on the Multiphor II unit side and thermostat
unit side respectively. The cooling plate tubings and thermostatic circulator
tubings can then be locked separately.
Two Multiphor II 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.
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4. Installation
16
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 illustrated. 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.
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4. Installation
17
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 buffer tank should be assembled with the four levelling feet.
Make sure the buffer tank 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 the anode electrode onto the buffer tank.
Connect the anode socket (red lead) to the anode pin on the right side of the
buffer tank.
Place a spirit level on the anode electrode and adjust the levelling feet until
the unit is levelled.
4.2 Multiphor II
NovaBlot Unit
for electro-
phoretic
transfer
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