Ettan IPGphor II

Ettan IPGphor II
user manual
2-D electrophoresis and first-dimension IEF
Ettan IPGphor II
isoelectric focusing system
um
80-6505-22/Rev. A0/06-03
Page finder
1. Ettan IPGphor II function and description
1.1.
1.2.
1.3.
1.4.
Unpacking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Instrument components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2
Important information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
2. Instrument set up
2.1. Placing the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
2.2. Power connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
2.3. Optional: Serial port connector for a printer or computer connection . . . . . . . . . . .7
3. Operating instructions
3.1. Power up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
3.2. Creating and editing protocol programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9
3.2.1. Protocol parameters
3.2.2. Ettan IPGphor II Control Software
3.2.3. Editing protocol parameters
3.3. IPG strip rehydration and sample application:
IPGphor Cup Loading Strip Holder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
3.4. IPG strip rehydration and sample application:
Fixed-length IPGphor Strip Holder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
3.5. Running a protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
3.5.1. Final assembly
3.5.2. Starting isoelectric focusing
3.5.3. Pausing or stopping a protocol
3.5.4. Printing a log of instrument conditions
4. Care and maintenance
4.1. Important! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
4.2. Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
4.3. Replacing fuses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
5. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
6. Customer service information
6.1. Technical service and repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
6.2. Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Protocol worksheet
....................................................................................
26
• pi
Ettan IPGphor II Isoelectric Focusing System • safety warnings and precautions
Safety warnings and precautions
English
Important user information
Please read this entire manual to fully understand the safe and effective use of this product.
The lightning symbol within an equilateral triangle is intended to alert the user to the risk of
exposure to high voltages. Should you have any comments on this manual, we will be pleased
to receive them via email at [email protected] or at:
Amersham Biosciences AB
SE-751 84
Uppsala, Sweden
Amersham Biosciences reserves the right to make changes in the specifications without
prior notice.
Warranty and Liability
Amersham Biosciences guarantees that the product delivered has been thoroughly tested to
ensure that it meets its published specifications. The warranty included in the conditions of
delivery is valid only if the product has been installed and used according to the instructions
supplied by Amersham Biosciences.
Amersham Biosciences shall in no event be liable for incidental or consequential damages,
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.
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 the company.
Renseignements importants d’utilization
Français
Pour une bonne compréhension et une utilisation en sécurité maximale, il convient de lire
entièrement ce manuel. Le symbole de l’éclair dans un triangle équilatéral a pour objet d’attirer l’attention de l’utilisateur sur un danger d’exposition à la haute tension. Tous vos
commentaires sur ce manuel seront les bienvenus et veuillez les adresser à:
Amersham Biosciences AB
SE-751 84
Uppsala, Sweden
Amersham Biosciences se réserve le droit d’effectuer des modifications de ces spécifications
sans aucun préavis.
Garantie et responsabilité
Amersham Biosciences garantit à l’utilisateur que le produit livré a subi avec succès tous les
essais prévus pour s’assurer qu’il est conforme aux spécifications et normes en vigueur. La
garantie incluse dans les conditions de livraison n’est valable que si le produit a été installé
et utilisé conformément aux instructions fournies par Amersham Biosciences.
La société Amersham Biosciences ne sera en aucun cas responsable de tout dommage causé
directement ou indirectement par toute utilisation incorrecte ou non approuvée du produit ou
découlant de cette utilisation, y compris toute perte de bénéfice ou de recettes, toute perte de
perspectives commerciales, tout empêchement d’utilisation et tout autre risques ayant un rapport
avec l’utilisation du produit, mais sans aucune limitation quant à la nature de ces dommages.
La reproduction, le stockage dans un système de récupération d’informations ou la transmission sous quelque forme que ce soit et par quelque moyen que ce soit de la présente publication en totalité ou en partie sont strictement interdits sans autorisation préalable écrite de
la société.
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• pii
• safety warnings and precautions
Información importante para el usuario
Para comprender el producto y utilizarlo con seguridad es necesario leer este manual en su
totalidad. El símbolo del rayo en un triángulo equilátero alerta al usuario sobre el riesgo de
exposición a altas tensiones. Si desearan hacer algún comentario sobre este manual, tengan
la amabilidad de remitirlo a:
Español
Amersham Biosciences AB
SE-751 84
Uppsala, Sweden
Amersham Biosciences se reserva el derecho a modificar las especificaciones sin previo aviso.
Garantía y responsabilidad
Amersham Biosciences garantiza que el producto entregado ha sido probado a fondo para
comprobar el cumplimiento de las especificaciones publicadas. La garantía incluida en las
condiciones de entrega sólo es válida si el producto se ha instalado y utilizado de acuerdo
con las instrucciones entregadas por Amersham Biosciences.
Amersham Biosciences no será responsable, bajo ningún concepto, de daños directos o indirectos, incluyendo sin limitación la pérdida de beneficios, la pérdida de ingresos, la pérdida de
oportunidades de negocio, la pérdida de utilización y otras consecuencias relacionadas,
cualquiera que sea la causa, que se deban a la utilización defectuosa e incorrecta del producto.
No está permitida la reproducción, ni el almacenaje en un sistema de recuperación, ni la
transmisión de parte alguna de esta publicación sin la autorización por escrito de la empresa.
Wichtige Benutzerinformationen
Deutsch
Für ein vollständiges Verständnis und eine sichere Handhabung dieses Produktes ist es
notwendig, daß der Benutzer dieses Handbuch vollständig durchliest. Ein Blitzsymbol in
einem gleichseitigen Dreieck soll den Benutzer auf die Gefahr anliegender Hochspannungen
hinweisen. Wenn Sie Anmerkungen zu diesem Handbuch haben, dann senden Sie diese
bitte an:
Amersham Biosciences AB
SE-751 84
Uppsala, Sweden
Amersham Biosciences behält sich das Recht vor, die Spezifikationen ohne vorhergehende
Ankündigung zu ändern.
Gewährleistung and Haftung
Amersham Biosciences garantiert, daß das gelieferte Produkt sorgfältig auf die Einhaltung der
veröffentlichten Spezifikationen getestet wurde. Die in den Lieferbedingungen näher
erläuterten Gewährleistungsansprüche gelten nur dann, wenn das Produkt gemäß den von
Amersham Biosciences gelieferten Anweisungen installiert und benutzt wurde.
Amersham Biosciences übernimmt keinerlei Haftung für Schäden oder Folgeschäden,
einschließlich, aber nicht begrenzt auf Gewinneinbußen, Einkommensverluste, entgangene
Geschäftsabschlüsse, Verlust der Gebrauchsfähigkeit oder andere Verluste, die wie auch immer
durch eine fehlerhafte oder unsachgemäße Verwendung des Produkts verursacht wurden.
Die vorliegende Veröffentlichung darf nur mit vorhergehender schriftlicher Genehmigung
durch das Unternehmen vervielfältigt, in einem Abrufsystem gespeichert oder in irgendeiner
Form oder mit irgendwelchen Mitteln übertragen werden.
• piii
Ettan IPGphor II Isoelectric Focusing System • safety warnings and precautions
Informazioni importanti per l’operatore
Per un utilizzo sicuro del prodotto, leggere attentamente l’intero contenuto del presente
manuale. Il simbolo del fulmine all’interno di un triangolo equilatero indica all’utente la
presenza di un rischio di esposizione ad alte tensioni. Si prega di inviare eventuali commenti
al presente manuale a:
Italiano
Amersham Biosciences AB
SE-751 84
Uppsala, Sweden
Amersham Biosciences si riserva il diritto di apportare modifiche ai dati tecnici senza preavviso.
Garanzia e responsabilitá
Amersham Biosciences garantisce che prima della consegna il prodotto è stato collaudato a
fondo per soddisfare i requisiti specificati. La garanzia inclusa nelle condizioni di consegna
risulta valida solamente se il prodotto è stato installato ed utilizzato nel rispetto delle
istruzioni fornite da Amersham Biosciences.
Amersham Biosciences non potrà essere ritenuta responsabile di incidenti o danni consequenziali, inclusi’ma non limitati’a perdite di profitti, mancato guadagno, perdite di affari,
difetti di funzionamento e relative esposizioni, dovuti ad un utilizzo non corretto del prodotto.
Nessuna parte della presente pubblicazione può essere riprodotta, conservata in sistemi di
gestione dati o trasmessa in alcun forma né per nessuno scopo senza autorizzazione scritta
del produttore.
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• piv
• Ettan IPGphor II function and description
1. Ettan IPGphor II Isoelectric Focusing System
function and description
The Ettan™ IPGphor™ II Isoelectric Focusing System performs first dimension isoelectric focusing (IEF) in 2-dimensional (2-D) electrophoresis.
The system comprises Immobiline™ DryStrip gel strips, which contain an
immobilized pH gradient (IPG); strip holders, which serve as both and
IEF chambers for the IPG strips; and the Ettan IPGphor II unit, which
includes a high-voltage DC power supply, solid state temperature control,
and can be programmed with up to ten user-defined IEF protocols.
Programmable functions include:
•
•
•
•
•
•
rehydration time,
platform temperature,
maximum current limit,
voltage change pattern,
voltage for each step in the isoelectric focusing program, and
step duration.
IPG strips are available in five lengths —7, 11, 13, 18, and 24 cm— and a
number of pH ranges, several linear and nonlinear (listed in section 6.2,
Ordering information). Two types of strip holders are available, a fixedlength strip holder and a variable-length cup loading strip holder. Once
the IPG strip is in place in a fixed-length strip holder, no further handling
is required. The holder is placed on the Ettan IPGphor II electrode platform for isoelectric focusing and the sample is introduced either during or
after the rehydration step. Up to 12 strip holders of the same size can be
accommodated for any one protocol.
The cup loading manifold accommodates 3 mm wide strips from 7 to 24 cm
long. After rehydration, strips are placed in the strip holder and the sample
cup positioned as desired for loading in a particular zone. Up to 9 cup
loading strip holders can be accommodated at one time.
1.1. Unpacking
Unwrap all packages carefully and compare contents with the packing
list, making sure all items arrived. If any part is missing, contact your
local Amersham Biosciences sales office. Inspect all components for
damage that may have occurred while the unit was in transit. If any part
appears damaged, contact the carrier immediately. Be sure to keep all
packing material for damage claims or to use should it become necessary
to return the unit.
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Ettan IPGphor II Isoelectric Focusing System • IPGphor II function and description
1.2. Instrument components
Ettan IPGphor II control software
(computer not included)
positive (anode)
electrode area
negative (cathode)
electrode area
IPGphor cup loading manifold
control panel
Five strip holder lengths are available —
7, 11, 13, 18, and 24 cm — one for each
IPG strip length.
Note: all strips (and holders) used at any
one time must be of the same length.
A cup loading strip holder that accomodates all strip lengths is also available.
Cover
Pressure
block
Base
Sample
application wells
The mains power switch is located on
the back panel on the left side.
Serial port (RS232) for a
connection to a PC or printer.
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• p2
• Ettan IPGphor II function and description
Control panel
LCD screen
The Ettan IPGphor II is controlled by a 7-key membrane keypad. Instrument status is indicated on the LCD screen.
The main screen is “Protocol #1, Name.” The default location of the cursor is always under the protocol number
“1.” Select any protocol by pressing the up and down arrow keys to display the number of the desired protocol. Once
the selected protocol is started, the LCD screen indicates the parameters of the step currently being carried out.
In “edit mode” the LCD displays the programmable parameters. Place the cursor in the field to be edited using the
right and left arrow keys and adjust the value by pressing the up and down arrow keys.
COMMAND keys
The EDIT key switches the display to “edit mode,” in which protocol steps can be programmed. (See section 3.2.)
Pressing the EDIT key in edit mode switches the display back to the identification screen for the protocol being edited.
The left arrow key moves the cursor to the left. Moving the cursor off the screen to the left repositions the cursor at
the left side of the next row. At the bottom row the cursor is repositioned at the left side of the top row.
The right arrow key moves the cursor to the right. Moving the cursor off the screen to the right repositions the cursor
at the left of the next row. At the bottom row the cursor is repositioned at the left side of the top row.
The up arrow key increases the value of the selected field or digit, and the down arrow key decreases the value of the
selected field or digit.
The START key begins the selected protocol.
The STOP key pauses the protocol. Pressing the STOP key a second time cancels the protocol in progress.
HV ON indicator lamp
Platform
Electrode areas
The HV ON lamp indicates that the high voltage power supply is on and isoelectric focusing is in progress. This lamp
is not illuminated during the rehydration phase of a protocol.
Up to 12 fixed-length strip holders of the same length or 9 variable-length cup loading strip holders can be placed on
the platform for rehydration and IEF. The embedded electrode areas provide the electrical connection between strip
holder electrodes and the integral power supply. Platform temperature is controlled by Peltier thermoelectric modules.
Both electrode areas are gold-plated copper. Placing a strip holder so that it bridges both electrode areas completes
the electrical circuit (once the safety lid is in place). Each holder must be oriented properly:
Negative (cathode) area: Place the flat end of the holder in the shorter electrode area, visually verifying that there is
metal-to-metal contact between the electrode on the bottom of the holder and the electrode area for the fixed length
strip holders.
Positive (anode) area: The pointed end of the holder points toward the back of the unit, and the electrode must
contact the longer electrode area; the position of the tip should correspond to the guidemarks on the edge of the
platform (7, 11, 13, 18, and 24 cm) for the fixed length strip holders.
Safety lid
The safety lid must be properly latched before power can be applied. The pressure pads in the lid secure the strip
holders. A minimum of two pads should contact the cover of each strip holder to ensure an electrical connection.
Strip holders
The strip holders and manifold are made of aluminum oxide ceramic of minimal thickness for efficient heat transfer.
This material is very brittle so the holders are fragile and must be handled with care. The ceramic is treated with a
special coating to minimize protein binding and must be cleaned only with the IPGphor Cleaning Solution supplied to
retain that coating.
Fixed-length strip holders
Each fixed-length strip holder base holds a single IPG strip throughout rehydration and isoelectric focusing. A matching length holder is available for each size strip. The holders, like IPG strips, have a flat and a pointed and. The
pointed end (“point is positive”) designates the anode, and thus the location of the acidic end of the immobilized
gradient. Embedded electrodes provide electrical connection between electrode areas and the gel.
Sample application wells
Each of the two wells can accept up to 15 µl of sample (7.5 µl on either side of the strip) in excess of the rehydration volume (i.e. 30 µl maximum for both wells). Introduce the sample through the wells if it is not included in the
rehydration solution.
Cover
The cover protects the IPG strip during rehydration, and the shape of the underside of the cover ensures electrical
continuity during IEF by pressing strip against the electrode while allowing electrolysis gases to escape. Rehydration
and separation progress can be monitored visually through the clear cover.
Cup loading manifold
The
The
The
The
cup loading manifold holds up to 12 IPG strips.
manifold allows for cup loading or bridge loading.
manifold allows the user to run strips that have been rehydrated into the IPG strip using the reswelling tray.
manifold itself is not used for rehydration.
Electrodes
The cup loading manifold has contact pins on the side of the electrodes.
These contact pins make contact with the power supply electrode areas when placed on the IPGphor
separation platform.
Sample cups
Sample cups can be placed almost anywhere along the length of the IPGphor strip holder that is not blocked by
a protrusion. For proper sealing of the cup to the gel, the feet of the sample cup must all rest on the bottom of
the channel.
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Ettan IPGphor II Isoelectric Focusing System • IPGphor II function and description
1.3. Important information
Instrument operation
English
• Caution! Ettan IPGphor II is a high voltage instrument that can cause fatal
electrical shock if the safety features are disabled. The safety lid must be
properly latched before starting a protocol.
• Exceeding the recommended current limit of 50 µA per IPG strip can cause
the strip to burn and may damage the instrument.
• During isoelectric focusing, do not lean on the safety lid, do not apply excess
pressure or uneven weight to the lid, and do not place any items on the lid.
Such pressure could cause arcing between the strip holder electrodes and the
electrode areas, damaging the instrument.
• Plug the instrument into a properly grounded outlet.
• Always disconnect the power cord before servicing.
• Ensure that the cooling vents at the front, back, and bottom of the instrument
are not obstructed.
• Operate Ettan IPGphor II on a level surface.
• Avoid spilling liquids on the body of the instrument.
• Avoid scratching the electrode areas.
IPG strips, strip holders, and reagents
• The strip holders and manifold trays are very brittle and fragile.
Handle carefully: Do not bump or drop.
• Always wear gloves when handling IPG strips and the equipment that comes in
contact with them. This will help minimize protein contamination which can
result in artifactual spots in the silver stained 2-D patterns.
• Clean strip holders and manifold with the strip holder cleaning solution
provided. Clean all other components that come in contact with the IPG
strip or the sample with a detergent designed for glassware. Rinse well with
distilled water.
• Use the appropriate rehydration volume for the IPG strip length.
• Solutions containing urea may be warmed briefly. Do not heat any solutions
containing urea above 30–40 ºC as isocyanate, a urea degradation product, will
carbamylate the proteins in the sample, thus changing their isoelectric points.
• All chemicals should be of the highest purity. Double distilled water should
be used.
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• p4
• Ettan IPGphor II function and description
Informations importantes
Précautions d’emploi
Français
• Attention! l’Ettan IPGphor II est un instrument à très haut voltage pouvant
causer un choc électrique mortel si les conditions de sécurité ne sont pas
respectées. Le couvercle de sécurité doit être convenablement verrouillé avant
de démarrer un protocole.
• Dépasser les limites de courant recommandées, 50 µA un bande IPG, peut
amener les bandes à brûler et endommager l’instrument!
• Durant l’isoélectrofocalisation, ne pas s’appuyer sur le couvercle de sécurité, ne
pas exercer de pression excessive ni de poids inutile sur le couvercle, et ne pas
placer d’objets sur le couvercle. Une telle pression pourrait provoquer un arc
électrique entre les électrodes des cassettes et la plate-forme de migration
porte-électrodes, abîmant l’instrument.
• Brancher l’instrument à une prise de terre appropriée.
• Toujours déconnecter le cordon d’alimentation avant la maintenance.
• S’assurer que les ventilations devant et à l’arrière de l’appareil ne sont pas
obstruées.
• Utiliser l’Ettan IPGphor II sur une surface plane.
• Eviter de renverser des liquides sur l’instrument.
• Eviter de rayer la plate-forme de migration porte-électrodes..
Mèches IPG, cassettes et réactifs
• Les cassettes sont très fragiles. Manipulez-les avec précaution: évitez de les
heurter ou de les laisser tomber.
• Toujours mettre des gants pour manipuler les bandes IPG ainsi que
l’équipement en contact avec celles-ci. Ceci aide à minimiser la contamination
protéique qui peut occasionner des spots artefactuels sur le gel 2D après la
coloration à l’argent.
• Nettoyer les sarcophages avec la solution dédiée à cet effet. Nettoyez tous les
composants qui rentrent en contact avec les bandes IPG ou l’échantillon, avec
un détergent pour matériel en verre. Bien rincer avec de l’eau distillée.
• Utilisez le volume approprié de réhydratation.
• Les solutions contenant de l’urée peuvent être chauffées brièvement. Ne pas
chauffer de solution contenant de l’urée à plus de 30–40 ºC car l’isocyanate,
un produit de dégradation de l’urée, produira une carbamylation des protéines
de l’échantillon, changeant ainsi leurs points isoélectriques.
• Tous les produits chimiques doivent être de la plus grande pureté. Veillez à
utiliser de l’eau doublement distillée.
• p5
Ettan IPGphor II Isoelectric Focusing System • IPGphor II function and description
1.4. Specifications
This declaration of conformity is only valid
for the instrument when it is:
• used in laboratory locations, and
• used as delivered from Amersham
Biosciences except for alterations described
in the User Manual.
Working surface
Electrode areas
Strip holder capacity
Platform temperature
Gold-plated copper
Up to 12 fixed-length, up one manifold tray
15–30 °C ± 2 °C
Strip holders
Material
Aluminum oxide ceramic base, acrylic cover,
polycarbonate, platinum on titanium
Holders for IPG strip lengths: 7, 11, 13, 18, and 24 cm
Cup loading manifold
For all strip lengths, cup and bridge loading
User interface
*High relative humidity may result in condensation forming
on the platform surface. Runs conducted at a platform
temperature of 20 °C and relative humidity less than 70%
will generally not cause condensation. Platform temperatures greater than 20 °C allow for higher relative humidity
without condensation. Environments for which condensation
occurs on the platform surface may vary. If condensation
occurs, the unit may require conditioned air to reduce
temperature and relative humidity.
Control panel
7-key membrane keypad.
Liquid crystal display (LCD)
4 lines, 24 characters per line
Programmable parameters
Rehydration time, platform temperature, maximum
current limit per strip, step voltage, step voltage
change pattern, and step duration.
Protocol capacity
10, with up to 9 steps each
Serial port
RS232C, 1200 or 9600 baud, 8 data bits
1 stop bit, no parity, no flow control
Operating environment
Temperature
Relative humidity
15–35 °C
0 –70%*
Power requirements
Line voltage
Frequency
Power consumption
115/230 V~
50/60 Hz
100 W
Power outputs
Voltage
Current
Power
—, resolution: 10 V
0–8000 V--0 –1.5 mA, resolution: 1 uA
12 W max.
Physical parameters
Dimensions (h × w × d)
Weight
14 × 25 × 46 cm
6.8 kg (15 lbs)
Safety
Safety features
Safety certifications
um 80-6505-22
• p6
Automatic voltage cutoff when safety lid is opened
CE, UL, CSA
• instrument set up
2. Instrument set up
2.1. Placing the instrument
1
Important! The vents at the front, back, and bottom of the instrument must
not be obstructed. Air must be free to circulate for the cooling apparatus to
function properly.
2
Place the instrument on a flat surface with the bubble level in the center.
Turn the leveling feet as necessary to make the instrument level and stable.
2.2. Power connection
Two power cords are supplied: one cord has a “North American” style (NEMA
5-15-P) 3-pronged plug for 115 V~ power outlets and the other has a “Central
European” style (CEE 7/7) plug for 230 V~ power outlets.
1
Select the cord with the appropriate plug for the power outlet in your laboratory.
If neither of these cords are suitable for your power outlets, obtain a detachable
cord with an IEC-320-C13 input.
2
Plug the connectors into the power cord receptacle on the rear panel and into a
properly grounded power outlet.
2.3. Optional: Serial port connector for a printer or
computer connection
The RS232C serial port for a printer or computer connection is at the left rear
of the unit. The type of cable required (straight serial or null modem) depends
on the type of device (DTE or DCE) that it is connected to: If the printer or
computer is configured to receive data on pin 2 and transmit on pin 3, a regular
straight serial cable is required. For any other configuration, a null modem cable
is required.
Pin number assignments and device set up details follow:
Ettan IPGphor II RS232C signal and pin number assignments:
Pin 2
Transmit
Pin 3
Receive
Pin 5
Ground
Other pins
Not connected
The Ettan IPGphor II requires these settings in the device receiving data:
Baud rate
Serial Port Setup
Baud Rate: 1200
Set Up or Dn
Exit>
1200 or 9600
Data bits
8
Stop bit
1
Start bit
1
Parity
None
Flow control
None
To set the baud rate Press the Up and Down arrows together to access the Baud
menu: Use the Up or Down arrows to set the baud rate and the Right arrow to
exit to the main menu.
• p7
Ettan IPGphor II Isoelectric Focusing System • operating instructions
3. Operating instructions
The following sections will guide you through each stage of the isoelectric
focusing process using the Ettan IPGphor II.
3.1. Power up
Turn on the mains power switch (|) located on the right side of the rear
panel. This activates a self diagnostic program that runs for approximately
10 seconds. A series of screens indicates progress. If any component fails,
the diagnostic program will stop and a message will indicate the source of
the fault; note the fault and press the START key to skip to the next test.
If any failure is detected, call the technical service department.
Once the diagnostic program is successfully completed, the main screen
indicates that the instrument is ready, as shown below.
Fig 1. Ettan IPGphor II control panel.
The main screen is “Protocol #1, Name.” The
default location of the cursor is always under
the protocol number “1.” Select any protocol
by pressing the up and down arrow keys to
display the number of the desired protocol.
Prot# 1 File 1
Rehydrate 0:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
The STOP key pauses the protocol.
Pressing the STOP key a second time
cancels the protocol in progress, and
pressing it a third time resets the
instrument to start another protocol.
The START key
begins the
selected protocol.
The up arrow key increases the value of the
selected field or digit, and the down arrow
key decreases the value of the selected
field or digit. During electrophoresis, the
up/down arrow keys are used to toggle
between VhS, VhT, and Hrs.
The left arrow key moves the
cursor to the left. Moving the
cursor off the screen to the left
wraps the cursor either to the
left end of the next line or to
the left end of the top row.
um 80-6505-22
• p8
The EDIT key switches the display to “edit
mode,” in which protocol steps can be
programmed. Pressing the EDIT key in edit
mode switches the display back to the identification screen for the protocol being edited.
The key also allows the estimated run time
remaining to be viewed while the unit
is operating.
The right arrow key moves the
cursor to the right. Moving the
cursor off the screen to the
right repositions the cursor at
the left of the next row or
wraps the cursor to the left
end of the top row.
• operating instructions
3.2. Creating and editing protocol programs
3.2.1. Protocol parameters
The Ettan IPGphor II can store up to ten protocols. Programmable
protocol parameters include: duration and temperature of the rehydration
phase; and duration, temperature, and power parameters for up to nine
IEF steps for each protocol. Programmable power parameters include
maximum current, voltage, and voltage change pattern.
Please refer to the 2-D Electrophoresis, principles and methods guide for
sample handling recommendations and protocol guidelines. Protocol
worksheets (pages 27–28) can aid in mapping out and documenting each
protocol. The allowable range of values for each programmable parameter is listed below.
Fig 2. Ettan IPGphor II Control Software
Parameter
operational range
Steps
1–9
recommended values
Step duration (hrs)
rehydration: 0–99:59
focusing: 0–99:59
10:00–14:00
2:00–10:00
Volts (V)
0–8000
100–8000
Max. current
1500 µA total for 12 strips max.
50 µA per strip
Platform temperature (°C)
rehydration: 15–30
focusing: 15–30
20
20
Navigating parameter “fields” and “digits.” Each screen consists of information as well as one or more fields that can be edited. The active fields
contain individually adjustable digits or characters. Certain digits are also
linked to their neighboring digits. For example, three of the four digits in
the voltage field can each be adjusted individually, but when adjusting the
value from 1000 to 990, for instance, even though only the ten’s place is
adjusted manually, three digits are affected. Whether individual or linked,
digits “wrap around,” so increasing from 9 produces 0 and decreasing
from 0 produces 9— until a parameter limit is reached. The
Ettan IPGphor II can be programmed in volt-hours (Vhr) or
hours and minutes in step-n-hold or in gradient mode. Any
values within the operating limits listed above can be used
3.2.2 Ettan IPGphor II Control Software
Ettan IPGphor II Control Software ( 80-6506-55) is available
which allows the user to control the instrument and capture
data from the serial port with a Windows™ based computer
and serial cable. The following functions are provided:
• Editing of the protocols, loading to and retrieving them
from the instrument.
• Starting, stopping and pausing of IEF runs on the instrument. Real time capture of a log file and graphing of the
voltage, current and volthours at user selected intervals
from 1–30 minutes.
• Printing and saving of the data to a text or spreadsheet file.
For detailed instructions on installation and usage of this
software, please refer to the Ettan IPGphor II Control
Software User Manual (80-6500-47).
• p9
Ettan IPGphor II Isoelectric Focusing System • operating instructions
3.2.3. Editing protocol parameters
All protocol parameters are set in the edit mode. Pressing the EDIT key
toggles between the first editable screen and the protocol identification
screen. For each protocol, the four different screens accessible in edit
mode are:
Screen 1. Rehydration and IEF parameters.
Screen 1
Protocol number and name
Rehydration time, temperature
IEF run conditions (temperature and maximum
current/strip), number of strips and number of steps in the
selected protocol.
Screen 2–4
the nine steps available for IEF conditions (voltage change
pattern, voltage, and step duration) in groups of 3
This section covers the programming, editing, or verifying of all
parameter values
1
Select the protocol to be created or edited from the main screen: Place the cursor
under the protocol number field using the right and left arrow keys and then move
to the desired protocol number using the up and down arrow keys.
Prot# 1 File 1
Rehydrate 0:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
The file name may be edited by using the right arrow to move into the file name
and up and down arrows to adjust the characters as desired. To skip editing the
name use the left arrow to proceed to the next line.
To change any letter, move the cursor to that location using the right and left
arrow keys and then scroll through the 37 available options using the up and
down arrow keys. Once the desired letter appears, move the cursor to the next
character to be set.
The original label “File #” occupies 6 of the 16 available characters. To change
the label to a more descriptive name, such as “11 CM 3–10L,” requires scrolling
through the alpha-numeric options for each of the 11 digits in the name.
2
Edit the Rehydration parameters, time and temperature and the IEF parameters,
temperature, uA/Strip and number of strips as necessary.
Prot# 1 File 1
Rehydrate 10:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
Note that the cursor stops only in the fields that can be edited. Once the cursor
is in place, press the up or down arrow keys to adjust the value.
The available time range is 0–99:59 h, with 10 min resolution. (Hence, only the
first three digits are editable.) A minimum of 10 h is required for the rehydration
of IPG strip gels, but overnight is recommended. This protocol step can also be
used as a simple timer to start the protocol at a convenient time. If the IPG strips
are already rehydrated and ready for immediate focusing, set the rehydration
duration to 00:00 Hrs.
um 80-6505-22
• p10
• operating instructions
To adjust the rehydration temperature, use the left and right arrow keys to place
the cursor and the up and down arrow keys to select the value within the range of
15–30 °C. The original factory programmed value is 20 °C.
Note: Maintaining the actual platform
temperature at <18 °C or >25 °C may require
placement of the Ettan IPGphor II unit in a
location of lower or higher than ambient
temperature (e.g. a cold room).
It may be necessary to operate the instrument
in an air conditioned room to prevent condensation from collecting on the platform in
humid environments when the platform is
run at greater than 7 °C below ambient
temperature.
Screen 1. rehydration and IEF parameters.
Prot# 1 File 1
Rehydrate 10:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
The third line contains fields to set the platform temperature and current limit for
all isoelectric focusing steps:
The original factory programmed IEF temperature is 20 °C. To adjust the temperature, use the left and right arrow keys to place the cursor in the active °C field,
and use the up and down arrow keys to select the value within the range of
15–30 °C.
Prot# 1 File 1
Rehydrate 10:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
The original factory programmed value is 50 µA per IPG strip. This is the maximum recommended setting to prevent overheating and possible damage to the
strip or the unit.
To adjust the current limit, use the left and right arrow keys to place the cursor
under one of the three active µA digits, and use the up and down arrow keys to
select the value within the range 0–200 µA per strip.
Note: The total deliverable amperage is 1500 µA.
Prot# 1 File 1 Rehydrate
10:00Hr @ 20°C
IEF @ 20°C 50uA/Strip
1 Strips 0 Steps
Use the arrow keys to adjust the numbers of strips to run.
To advance to the next edit screen, press the EDIT key.
3
Screens 2–4. IEF protocol steps.
The second screen encountered in edit mode is the first in a series of nine
programmable IEF steps in which the voltage change pattern, voltage, and step
duration are set. The steps are displayed in groups of 3 steps per screen.
The number of steps which have been programmed is displayed next to the
protocol number.
Each step is identified by “S” and the step number and can be accessed by
placing the cursor under the step number using the right and left arrow keys.
To advance to the next step group press the down arrow key and to return to a
previous step group press the up arrow key.
• p11
Ettan IPGphor II Isoelectric Focusing System • operating instructions
The two options for the voltage change pattern are: “Step,” which sets the voltage
at the selected value for the new step and then holds the voltage constant for the
step duration; and “Gradient,” which increases the voltage limit (linearly with
respect to time) from the value set for the previous step to the value set for this
step. If this is the first step, the value increases from 0.
The original programmed option is “Step” for each step. To toggle to “Gradient,”
move the cursor to the Step field using the right and left arrow keys and toggle
with the up or down arrow key:
Prot#1 3 Steps
S1 Grad 500V 1:00 Hrs
S2 Grad 1000V 4:00 Hrs
S3 Step 8000V 80000 Vhr
IEF voltage is programmable from 0 –8000 V in increments of 10 V. Each of the
first three digits in the voltage field can be set independently. First move the
cursor to the desired digit location using the right and left arrow keys and then
change the value using the up and down arrow keys.
Prot#1 3 Steps
S1 Grad 500V 1:00 Hrs
S2 Grad 1000V 4:00 Hrs
S3 Step 8000V 80000 Vhr
The original factory programmed voltage for each step is 0. Program each step
with the protocol voltage values. If fewer than nine steps are required, end the
program by assigning a value of 0 V after the final step. The step duration (0 to
99:59 hours) or volt-hours (0 to 300000 Vhr) are programmable for each step.
To select, set the cursor under the currently selected field and use the up or down
arrow key to toggle between Hrs and Vhrs. To set the digits, first move the cursor
to the desired digit location using the right and left arrow keys and then change
the value using the up and down arrow keys.
Note: When toggling from Hrs to Vhr, the
value in the Vhrs field is automatically
calculated according to the selected voltage
change pattern:
For Step-n-hold, Vhr is the product of hours
and voltage.
For Gradient, Vhr is the product of hours and
the mean of the voltage limit set for the
current step and the previous step.
Absolute Vhr limits can be set to any value
from 1–30000 Vhr.
um 80-6505-22
• p12
The original factory programmed time for each step is 0:00, and should be set
to 0:00 for all unused steps. The Vhrs value is calculated according to the
explanatory note at the left, but these values can be set in the same manner as
the Hrs values.
Prot#1 3 Steps
S1 Grad 500V 1:00 Hrs
S2 Grad 1000V 4:00 Hrs
S3 Step 8000V 80000 Vhr
hours step
volt-hours step
• operating instructions
Programming example
Simplified 4-step protocol and resulting voltage profile
Prot#1 3 Steps
S1 Grad 500V 1:00 Hrs
S2 Grad 2000V 1:00 Hrs
S3 Step 8000V 2:00 Hrs
To illustrate the two voltage change patterns, a
simplified example shows program steps and the
resulting voltage profile. Note that step 4 ends
the protocol.
For recommended IEF protocols, see the 2-D
Electrophoresis Principles and Methods guide.
Fig 3. Voltage profile.
The set voltage is the voltage limit. Under conditions
of limiting current, it may take some time for the
maximum voltage to be reached, and under certain
power conditions, it may not be reached at all.
A lower voltage step may be entered at the end of the Ettan IPGphor II protocol.
Band sharpness will decrease however.
4
To verify the protocol, scroll through each step: place the cursor in the step
number field using the right and left arrow keys and then advance through the
steps using the up and down arrow keys.
Note: Pressing the up arrow key in screen 4
wraps back to the rehydration screen.
Edit as required by placing the cursor with the right and left arrow keys and
adjusting the value with the up and down arrow keys. The Protocol Worksheets at
the back of the manual can be useful in this step.
Make sure that the voltage field in all unused steps displays a value of 0, or that
the value in the Hrs field is 00:00.
5
Press the EDIT key to save any changes and exit the step edit mode. The protocol
identification screen will appear.
• p13
Ettan IPGphor II Isoelectric Focusing System • operating instructions
3.3. IPG strip rehydration and sample application:
Manifold Cup Loading Tray
Ettan IPGphor II Cup Loading Manifold
The Ettan IPGphor II Cup Loading Manifold is suitable for first-dimension
isoelectric focusing (IEF) of proteins on Immobiline DryStrip immobilized
pH gradient (IPG) gel strips 3 mm wide, from 7 cm to 24 cm long, in
the Amersham Biosciences Ettan IPGphor II Electrophoresis unit. IEF is
performed with the gel side facing up. Samples are applied in a localized
region through an open bottom loading cup. Cup loading has been found
to improve protein focusing patterns, particularly on basic IPG strips
(pH 6–9 and 6–11). Under conditions where substantial water transport
(electroendosmosis) accompanies focusing, such as with protein loads in
excess of 1 mg, the face up mode frequently yields better resolution. The
Manifold can accommodate anodic and cathodic loading.
Fig 4. Components of the Ettan IPGphor II
Cup Loading Manifold system.
B.
E.
H.
A.
D.
G.
J.
C.
F.
I.
A.
B.
C.
D.
E.
F.
um 80-6505-22
• p14
Manifold ceramic tray
Pre-cut electrode wicks
Lid adapter
Electrode assembly
Sample cups
Cleaning brush
K.
G.
H.
I.
J.
K.
Pre-cut paper bridges
Sample cup insertion tool
Cleaning solution
Forceps
Spirit level
• operating instructions
3.4. IPG strip rehydration and sample application:
Fixed-length IPGphor Strip Holders
IPGphor fixed-length strip holders allow IPG strips to be rehydrated and
samples loaded in one step before proceeding automatically to perform
the separation. The IPG strips are 3 mm wide and 0.5 mm thick after
rehydration. To rehydrate:
1
Important! Handle the holders with care, as
they are brittle and fragile.
Prepare the strip holder(s).
Select the strip holder(s) corresponding to the IPG strip length chosen for the
experiment (7, 11, 13, 18, or 24 cm). Wash each holder with strip holder cleaning
solution supplied to remove residual protein. Rinse thoroughly with double distilled
water. Use a cotton swab or a lint-free tissue to dry the holder or allow it to air-dry.
The holder must be completely dry before use.
2
Prepare the rehydration solution
The 2-D Electrophoresis, Principles and Methods guide contains commonly used
recipes. Required volumes per strip are itemized in the table below:
IPG strip length
(cm)
Total volume per strip holder
(µl, including any sample)
7
125
11
200
13
250
18
350
24
450
3
Pipet the appropriate volume of rehydration
solution into each holder.
Important! To ensure complete sample uptake,
do not apply excess rehydration solution.
Deliver the solution slowly at a central point in
the strip holder channel away from the sample
application wells. Remove any larger bubbles.
4
Place the IPG strip
Remove the protective cover from the IPG strip.
Position it with the gel side down and the pointed
(anodic) end of the strip directed toward the
pointed end of the strip holder. Pointed end first,
lower the strip onto the solution. To help coat the
entire strip, gently lift and lower the strip and
slide it back and forth along the surface of the
solution, tilting the strip holder slightly as needed
to assure complete and even wetting.
Finally, lower the cathodic (square) end of the
strip into the channel, making sure that the IPG
gel contacts the strip holder electrodes at each
end. (The gel can be visually identified once the
rehydration solution begins to dye the gel.) Be
careful not to trap bubbles under the strip.
• p15
Ettan IPGphor II Isoelectric Focusing System • operating instructions
5
Apply Immobiline DryStrip Cover Fluid to minimize evaporation and urea crystallization. Pipet the fluid dropwise into one end of the strip holder until one half of
the strip is covered. Then pipet the fluid dropwise into the other end of the strip
holder, adding fluid until the entire IPG strip is covered.
6
Place the cover on the holder. Pressure blocks on the underside of the cover assure
that the strip maintains good contact with the electrodes as the gel rehydrates.
7
Rehydration can proceed on the bench top or on the Ettan IPGphor II unit platform.
Ensure that the holder is on a level surface. A minimum of 10 h is required for
rehydration; overnight is recommended. Alternatively, the rehydration period can
be programmed as the first step of an Ettan IPGphor II protocol. This is especially
convenient if temperature control during rehydration is a concern, or if a low
voltage is applied during rehydration.
Optional: Sample rehydration under voltage
If desired, the sample can be absorbed by the IPG strip under low voltage. This
may improve the uptake of high molecular weight proteins. To perform rehydration
under voltage, set the rehydration time to 0:00 and program Step 1 of the protocol for low voltage (30 –100 V) for 10–12 h. Program additional steps as desired
to achieve complete focusing.
Optional: Apply electrode pads just prior to IEF
Under certain conditions, such as prolonged focusing, water may migrate toward one
end of the strip, causing the other end to begin drying out. This effect can be minimized by placing paper electrode pads between the IPG strip and each strip holder
electrode just before IEF. Electrode pads may also absorb ions that would otherwise
accumulate at the ends of the IPG strip and possibly interfere with the separation.
1
To apply, cut two 3 mm wide electrode pads from a paper IEF electrode strip
(18-1004-40). Place on a clean, flat surface such as a glass plate and soak
with deionized water. Remove excess water by blotting with tissue paper.
Important! Electrode pads must be damp, not wet.
2
Using forceps or tweezers, lift one end of the rehydrated IPG strip. Position an
electrode pad over the electrode, then lower the strip back in place. Repeat at
the other end.
Optional: Apply sample after rehydration
If the sample was not applied by inclusion in the
rehydration solution, it can be applied immediately prior to IEF.
Note: The IPG strip backing is impermeable;
do not apply the sample to the back of the strip.
1
Prepare the sample in a solution similar in
composition to the rehydration solution used.
2
Pipet the sample into either or both of the lateral
wells at either end of the strip holder. Introduce
the sample below the cover fluid.
Up to 7.5 µl of sample solution can be added to
each side (i.e. 15 µl per well or 30 µl max. if
both sides of both wells are used).
um 80-6505-22
• p16
• operating instructions
3.5. Running a protocol
3.5.1. Final assembly
1
Place the strip holders
The Ettan IPGphor II platform has two electrode areas: the larger area is the positive electrode (anode) and the smaller area is the negative electrode (cathode).
Position the strip holder on the platform as shown below: The pointed end of the
holder is over the anode (pointing to the back of the unit) and the blunt end is
over the cathode. Guidemarks along the sides of the platform show approximate
positioning for each strip holder size (7, 11, 13, 18, and 24 cm). Check that each
of the two external electrode contacts makes metal to metal contact.
the pointed end of the strip holder
is in the anodic electrode area.
external electrode contacts
(2, underneath, one in
each electrode area)
anodic electrode area (+)
cathodic electrode area (–)
2
Close the safety lid
If using fixed length strip holders place the Lid adapter (80-6499-71) on top of
the strip holders with the bent edge down at the front of the unit.
• p17
Ettan IPGphor II Isoelectric Focusing System • operating instructions
3.5.2. Starting isoelectric focusing
1
Advance to the protocol identification screen by placing the cursor in the protocol
number field using the right and left arrow keys and selecting the protocol
number using the up and down arrow keys.
Prot# 1 MY PROTOCOL
Rehydrate 10:00Hr @ 20°C
IEF @ 20°C 200uA/Strip
12 Strips 3 Steps
2
Use the right and left arrow keys to position the cursor to set the number of strips
to run, and the up and down arrow keys to select the number (1–12):
Press the START key.
A screen is displayed to confirm the number of strips to run. Adjust the number
of strips if necessary. Press START to continue.
Number of strips :12
Press START to continue
If the safety lid is not properly latched the following message will indicate the
required action:
Important! During IEF:
Lid open step 1
Close lid to continue
• do not lean on the safety lid,
• do not apply excess pressure or uneven
weight to the lid, and
• do not place any items on the lid.
Important! Pressure on the safety lid could
cause arcing between the strip holder electrodes and the electrode areas, and may
damage the instrument.
After securing the safety lid, press the START key again.
Once the protocol begins, each step is carried out automatically, and instrument
status for each step is indicated by the screen.
Platform temperature and time remaining can be changed while the protocol is in
progress, and each such possibility is discussed in turn below.
The first screen will indicate the protocol, number of steps, and total hours
(excluding rehydration time):
Starting protocol # 1
3 steps 4:00 Hr
If a rehydration period was programmed, the rehydration status screen will
indicate the number of hours elapsed and the actual platform temperature:
Running Prot# 1
Rehydration for 10:00 Hr
0:01 Hr Elapsed
Rehydrate at 20°C
um 80-6505-22
• p18
• operating instructions
To change rehydration duration or other parameters, press the EDIT key to display
the set screen:
The first line indicates the protocol is running. Pressing Edit again will access the
step setting screen. Pressing Edit again will return to the Running screen. Any
changes made while a protocol is running are applied to the current run only, the
stored protocol is not affected.
Running Prot# 1
Rehydrate 10:00Hr @ 20°C
IEF @ 20°C 200uA/Strip
12 Strips 3 Steps
Move the cursor under the digit you wish to set and change it using the up and
down arrow keys. To end this step, set all digits to 0. This screen is automatically
replaced by the regular status screen after a few seconds. The temperature field
can be changed directly (without pressing the EDIT key): use the right and left
arrow keys to place the cursor in the temperature field and use the up and down
arrow keys to adjust the value. It may take up to 30 minutes to reach the set
temperature, depending on ambient conditions.
Isoelectric focusing will begin after the rehydration step is complete. The HV ON
lamp will light and the status screen showing the step currently in progress as
well as all parameters will appear:
Note: The indicated wattage is the total
power delivered to all strips, and will often
read 0 W because any value less than 0.5 is
rounded down to 0. If the delivered power
exceeds 0.4 W per strip, stop the run and
reduce the V or µA settings.
Running Prot# 1 @ 20°C
Step 1 of 3 Vhrs
Step Vhrs Elapsed
500V 40uA/Strip 0.0W
The actual platform temperature is indicated.
The average current per IPG strip (µA), the total instantaneous power delivered
(W), and actual voltage (V) are indicated. If the current is the limiting parameter,
the “A” in “µA” will flash.
The Ettan IPGphor II will display the volt-hours during the run as VhS, which
indicates the total volt hours for that step, or VhT, the total volt-hours accumulated during the entire run at the current time. Hours elapsed for the current step
will be indicated by Hrs. To toggle between VhT, VhS and Hrs, move the cursor to
the Hrs/Vhrs field of the display using the right or left arrow key and change the
display to reflect VhT, VhS and Hrs using the up and down keys.
To change the display to show remaining Hrs, VhS or VhT move the cursor under
the Elapsed or ~ Remain field and use the up or down buttons.
The temperature, voltage and current limits for the current step may be adjusted
directly on this screen as well.
Pressing the Edit key will access the step parameter screen. Pressing Edit again
will return to the Running screen. Use the up arrow in the S1 field to access the
Rehydration/IEF screen.
Running
S1 Grad
S2 Grad
S3 Step
Prot#
500V 1:00 Hrs
2000V 1:00 Hrs
8000V 2:00 Hrs
Note: If you adjust the voltage during a gradient step the step will be converted to
Step for the remainder of the step.
• p19
Ettan IPGphor II Isoelectric Focusing System • operating instructions
As isoelectric focusing proceeds, the bromophenol blue tracking dye will migrate
toward the anode and leave the IPG strip. This generally occurs well before focusing is complete. Note: A colorless strip is no indication that IEF is complete.
If the tracking dye does not move, then no current is flowing across the strip.
Check that the strip holder electrodes are in contact with the electrode areas.
See the Troubleshooting section for additional suggestions.
When the protocol ends, audible beeps will sound and the following screen
will display:
Run ended at
8000V 12000 Vhrs Total
50uA 8:00 Hr Total
Press Stop to reset
The final instrument values are displayed. Press the STOP key, as indicated,
to reset the instrument.
3.5.3. Pausing or stopping a protocol
To pause or stop a protocol in progress, press the STOP key:
Paused in step 1
Press START to continue
To continue the protocol, press the START key. To cancel the protocol, press the
STOP key again. This will cause the instrument to respond as though the end of
an uninterrupted protocol had been reached as described above.
3.5.4. Printing a log of instrument conditions
The RS232C serial port is programmed to report instrument status every
5 minutes during isoelectric focusing. The rehydration period is not logged.
Indicated are the time elapsed, voltage, current, and cumulative volt-hours.
Also indicated are any interruptions in power, such as when the lid is opened or
when the end of the programmed run is reached.
Amersham Biosciences
Ettan IPGphor II
Version A 05/25/03
Serial No. 20076543
An abbreviated log illustrates the format.
Protocol: NAME
No. of strips: 1
IEF Params: 50 µA/ strip at 20 C
S1 Step-n-hold 500V 3:00Hrs
0:00
50V at 1 µA
0Vhr
0:05
500V at 9 µA
37Vhrs
0:10
500V at 10 µA 78Vhrs
0:15
500V at 10 µA 120Vhrs
0:20
500V at 10 µA 162Vhrs
0:25
500V at 9 µA
0:26
***End of run ***
um 80-6505-22
• p20
204Vhrs
Ettan IPGphor II Control Software (80-6506-55) may also be used to log and
graph and print the output.
Additionally the output may be export to a text or spreadsheet file.
• care and maintenance
4. Care and maintenance
4.1. Important!
• Strip holders are made of ceramic and thus are very brittle and fragile.
Handle carefully. Do not bump or drop. When cleaning, take care not
to bend or break the strip holder electrodes.
• Take care to avoid scratching the electrode areas.
• Wipe the exterior surfaces with a damp cloth after each use. Never use
abrasive cleansers or solvents.
• Always turn the mains power switch off and unplug the power cord
before doing any maintenance.
4.2. Cleaning
The parts of the instrument that come in contact with liquid reagents are
resistant to chemicals typically used for IEF. Before introducing any other
chemicals into the system, first test the affected parts. Never use ketones,
hot strong acids, or hydrocarbon solvents.
IPGphor Strip Holders and manifold are made from a thermally conductive aluminum oxide ceramic. They have been surface-treated to minimize
adsorption and interaction with proteins. The surface coating is mechanically robust and can withstand temperatures of up to 300 ºC, however it
can be damaged by prolonged exposure to extremes of pH.
IPGphor Strip Holder Cleaning Solution has been specifically formulated
for removing protein deposits. It will not damage the strip holder or
manifold. IPGphor Strip Holder Cleaning Solution (80-6452-78) can be
ordered in 950-ml bottles from Amersham Biosciences.
Important! You must use a neutral pH
detergent, such as the IPGphor Strip Holder
Cleaning Solution, to remove residual protein
from these strip holders.
Important! Fixed length strip holders may
be baked, boiled or autoclaved. DO NOT
EXPOSE THEM TO STRONG ACIDS OR
BASES, INCLUDING ALKALINE DETERGENTS. THE CUP LOADING MANIFOLD
SHOULD NOT BE BAKED, BOILED OR
AUTOCLAVED!
Cleaning procedure
Clean strip holders after each first-dimension IEF run. Do not let solutions dry in the strip holder. Cleaning may be more effective if the strip
holders are first soaked a few hours to overnight in a solution of 2–5%
IPGphor Strip Holder Cleaning Solution in water. Strip holders may also
be immersed in boiling 1% SDS.
1
First rinse off the strip holder. Use a mild liquid soap to remove any residual
IPG cover fluid.
2
Squeeze a few drops of Ettan IPGphor II Strip Holder Cleaning Solution into the
strip holder slot. Use a toothbrush and vigorous agitation to clean the strip holder.
3
Rinse well with distilled or deionized water.
Thoroughly air dry the strip holders or dry well with a lint-free tissue prior to use.
Wipe the platform and plate electrodes with a lab tissue or paper towel after
each use. Other exterior surfaces can be cleaned with a damp cloth. Never use
abrasive cleanser or solvents.
• p21
Ettan IPGphor II Isoelectric Focusing System • care and maintenance
4.3. Replacing fuses
insert screwdriver in this notch to
open the cover.
Important! Fuses protect equipment by
disconnecting loads too large for the instrument’s circuit design, so it is imperative that
fuses are replaced only with fuses of identical
rating. The mains power module, located at
the back of the instrument, contains two
input fuses which are the same for all
models: T 1.6A 250V, 5 × 20 mm.
insert the screwdriver
blade behind the arrow
to pull the cassette
completely out.
mains power switch
hinged cover
Fig 4. The mains power module is located on the
back panel.
1
Caution! Turn the mains power supply switch off and detach the power cord
before replacing input fuses!
2
Open the fuse compartment by inserting a small flat-blade screwdriver into the
slot at the top of the power module. Twist the screwdriver 1/8 – turn to release the
cover, then pull out the hinged compartment, which opens out.
3
Insert the screwdriver above the arrow on one fuse cassette, catch the cassette
end, and slowly slide it completely out of the module.
4
Pull the fuse out of its cassette and inspect. If the fuse element is burned or
broken, replace the fuse with an identical type. If the fuse appears to be intact,
check it with a multi-meter. (A reading of 1 ohm or less indicates the fuse is
still usable.)
5
After placing a good fuse into the cassette, slide it into the power module,
making sure the arrow on the cassette points to the right (in the same direction
as the guide arrows on the inside of the compartment door).
6
Repeat steps 3 to 5 for second cassette.
7
Close the fuse compartment cover and gently press it into the power module until
it snaps shut.
8
Plug the power cord into the unit and turn the mains power switch on.
um 80-6505-22
• p22
• troubleshooting
5. Troubleshooting
symptom
possible solutions
Problems indicated by LCD messages
Lid open step 1, close to continue
The safety lid is not properly closed. When the safety lid is open, the system has an
automatic voltage cutoff safety feature. In order for the protocol to proceed, the safety lid
must be closed.
Locked screen in edit mode
Turn off the mains power switch to reset the instrument.
Blank display
If no electrical components are functioning (e.g. HV lamp does not light and the cooling fans
are motionless), check the fuses in the mains power module.
Diagnostic program indicates component failure
Note the component that failed and press the START key to continue through the diagnostic
program. Call your local Amersham Biosciences sales office for further information on how to
remedy the failure.
Power delivery
Current too low or zero
At least two of three pressure pads under the safety lid should press gently against the strip
holders to ensure electrical continuity between the strip holder electrodes and the electrode
areas on the platform.
The gel must be evenly and completely rehydrated to conduct current. Make sure the proper
amount of rehydration solution is applied to the IPG strip holder and allow a minimum of
10 hours for rehydration.
Voltage limit not reached
The ionic strength of the rehydration solution is too high; reduce the IPG buffer concentration; use a mixed-bed ion-exchange resin to remove ionic breakdown products of urea of
other additives.
Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM.
Sparks or burning in strips
Reduce the current limit. Do not exceed 50 µA per strip.
Prevent the IPG strip from drying out by always applying Immobiline DryStrip Cover Fluid
during rehydration.
Ensure that the IPG strip is fully rehydrated along the entire length of the strip. The IPG strip
should be in complete contact with the correct volume of rehydration solution. Remove any
air bubbles trapped under the IPG strip.
Desalt the sample or prepare the sample so that the salt concentration is less than 10 mM.
De-ionize additives to the rehydration solution. Excessive charged material in the sample or
rehydration buffer lead to electroendosmosis which will dry out the middle of the strip,
possibly leading to arcing and burning there.
• p23
Ettan IPGphor II Isoelectric Focusing System • customer service information
6. Customer service information
6.1. Technical service and repair
Important! Request a copy of the Amersham
Biosciences “Health and Safety Declaration”
form before returning the item. No items can
be accepted for servicing or return unless this
form is properly completed.
Amersham Biosciences offers complete technical support for all our
products. If you have any questions about how to use this product,
or would like to arrange to repair it, please call or fax your local
Amersham Biosciences representative.
6.2. Ordering information
product
quantity
code number
Ettan IPGphor II Isoelectric Focusing Unit
Basic unit. Order strip holders separately
1
80-6505-03
Fuses, 1.6 A, 250V, 5 × 20mm
5
80-6108-69
Cable Serial 9 Pin F/F with Null Modem Adapter
1
80-6506-36
Ettan IPGphor II Control software
1
80-6506-55
7 cm
2
80-6455-44
11 cm
2
80-6455-63
13 cm
2
80-6455-82
18 cm
2
80-6456-01
24 cm
2
80-6479-76
7 cm, complete
6
80-6416-11
11 cm, complete
6
80-6416-30
13 cm, complete
6
80-6416-49
18 cm, complete
6
80-6416-68
24 cm, complete
6
80-6469-88
7 cm, complete
1
80-6416-87
11 cm, complete
1
80-6417-06
13 cm, complete
1
80-6417-25
18 cm, complete
1
80-6417-44
24 cm, complete
1
80-6470-07
Manifold Kit
1
80-6498-38
7–18 cm
1
80-6371-84
7–24 cm
1
80-6465-32
Strip Holder Covers
Strip Holders for use with Immobiline DryStrip gels
Immobiline DryStrip Reswelling Trays
IPG Buffer
pH 4–7
1 ml
17-6000-86
pH 3–10 L
1 ml
17-6000-87
pH 3–10 NL
1 ml
17-6000-88
pH 3.5–5.0
1 ml
17-6002-02
pH 4.5–5.5
1 ml
17-6002-04
pH 5.0–6.7
1 ml
17-6002-05
pH 5.5–6.7
1 ml
17-6002-06
pH 6–11
1 ml
17-6001-78
DeStreak Rehydration Solution
1 ml
17-6003-19
DeStreak Reagent
5 × 3 ml
17-6003-18
Rehydration Solutions
um 80-6505-22
• p24
• customer service information
product
quantity
code number
Ettan preparation kits and reagents
2-D Fractionation Kit
10 preps
80-6501-04
Albumin and 1gG Removal Kit
10 preps
RPN6300
Protease Inhibitor
1 ml
80-6501-23
Nuclease Mix
0.5 ml
80-6501-42
Thiourea
100 g
RPN6301
Iodoacetamide
25 g
RPN6302
Sample Grinding Kit
50 samples
80-6483-37
2-D Clean-Up Kit
50 samples
80-6484-51
SDS-PAGE Clean-Up Kit
50 samples
80-6484-70
2-D Quant Kit
500 assays
80-6483-56
Mini Dialysis Kit, 1 kDa cut-off, 250 µl
1
80-6483-75
Mini Dialysis Kit, 1 kDa cut-off, 2 ml
1
80-6483-94
Mini Dialysis Kit, 8 kDa cut-off, 250 µl
1
80-6484-13
Mini Dialysis Kit, 8 kDa cut-off, 2 ml
1
80-6484-32
500 g
1g
17-1319-01
17-1314-01
PlusOne™ reagents
Urea
CHAPS
Triton™ X-100
500 ml
17-1315-01
Dithiothreitol (DTT)
1g
17-1318-01
Bromophenol Blue
10 g
17-1329-01
Immobiline DryStrip Cover Fluid
1000 ml
17-1335-01
Immobiline DryStrip gels
7 cm
pH 4–7
12
17-6001-10
pH 3–10
12
17-6001-11
pH 3–10NL
12
17-6001-12
pH 6–11
12
17-6001-94
11 cm
pH 4–7
12
18-1016-60
pH 3–10
12
18-1016-61
pH 6–11
12
17-6001-95
13 cm
pH 4–7
12
17-6001-13
pH 3–10
12
17-6001-14
pH 3–10NL
12
17-6001-15
pH 6–11
12
17-6001-96
18 cm
pH 4–7
12
17-1233-01
pH 3–10
12
17-1234-01
pH 3–10NL
12
17-1235-01
pH 3.5–4.5
12
17-6001-83
pH 4.0–5.0
12
17-6001-84
pH 4.5–5.5
12
17-6001-85
pH 5.0–6.0
12
17-6001-86
pH 5.5–6.7
12
17-6001-87
pH 6–9
12
17-6001-88
pH 6–11
12
17-6001-97
• p25
Ettan IPGphor II Isoelectric Focusing System • customer service information
product
quantity
code number
pH 3.5–4.5
12
17-6002-38
pH 4.0–5.0
12
17-6002-39
pH 4.5–5.5
12
17-6002-40
pH 5.0–6.0
12
17-6002-41
pH 5.5–6.7
12
17-6002-42
pH 3–7 NL
12
17-6002-43
pH 3–10
12
17-6002-44
pH 3–10 NL
12
17-6002-45
pH 4–7
12
17-6002-46
pH 6–9
12
17-6002-47
Cleaning solution, IPGphor Strip Holder
950 ml
80-6452-78
2-D Electrophoresis Principles and Methods
1
80-6429-60
24 cm
um 80-6505-22
• p26
Equilibration tube set for 24 cm IPG Strips
12
80-6467-79
IEF electrode strips
100
18-1004-40
Ettan IPGphor II Isoelectric Focusing System
First Dimension Protocol Worksheet
Program steps
IPG strip preparation
Prot# _ ________________
Rehydrate __:__Hr @ __°C
IEF @ __°C ___uA/Strip
__ Strips ? Steps
Prot#? ? Steps
S1 ____ ____V _____ ___
S2 ____ ____V _____ ___ S3
____ ____V _____ ___
Rehydration volume (circle one)
IPG strip length
(cm)
rehydration volume
per strip (µl)
7
125
11
200
13
250
18
350
24
450
Rehydration solution
reagent
final conc.
Urea
Detergent
Reductant
IPG buffer pH
Prot#? ? Steps
S4 ____ ____V _____ ___
S5 ____ ____V _____ ___ S6
____ ____V _____ ___
Dye
Sample
Water
Total volume
Notes:
Prot#? ? Steps
S7 ____ ____V _____ ___
S8 ____ ____V _____ ___ S9
____ ____V _____ ___
Step of Grad
_:_ Hrs or _____Vhrs
Date
Researcher
Sample
volume
or weight
Ettan, Immobiline, IPGphor, and PlusOne are trademarks of Amersham Biosciences Limited.
Amersham and Amersham Biosciences are trademarks
of Amersham plc.
Triton is a trademark of Union Chemicals and
Plastics Co.
Windows is a trademark of Microsoft Corporation.
© Amersham Biosciences Corp. 2003—
All rights reserved.
All goods and services are sold subject to the terms
and conditions of sale of the company within the
Amersham Biosciences group that supplies them.
A copy of these terms and conditions is available
on request.
Printed in the USA
www.amershambiosciences.com
Amersham Biosciences UK Limited
Amersham Place, Little Chalfont,
Buckinghamshire, England HP7 9NA
Amersham Biosciences AB
SE-751, 84 Uppsala, Sweden
Amersham Biosciences Corp.
800 Centennial Avenue, PO Box 1327,
Piscataway, NJ 08855 USA
Amersham Biosciences Europe GmbH
Munzinger Strasse 9, D-79111, Freiburg, Germany
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