Ettan IPGphor Cup Loading Manifold

Ettan IPGphor Cup Loading Manifold
user manual
2-D electrophoresis
Ettan IPGphor Cup
Loading Manifold
Ettan IPGphor Isoelectric Focusing System
um
80-6499-52/ Rev. A0/ 06-03
Page finder
Ettan IPGphor Cup Loading Manifold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
IPGphor unit modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Instructions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Suggested run conditions
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
Paper-bridge loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12
Care and cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Customer service information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Ordering information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
• pi
Ettan IPGphor Cup Loading Manifold • 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 exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the
instrument. If you have any comments on this manual, please send them to us 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 or incorrect use of the product. © 2002 Amersham
Biosciences Corp. 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 the company.
Renseignements importants d’utilization
Français
Pour une bonne compréhension et une utilisation en sécurité maximale, il convient de lire
entièrement ce manuel. Dans la documentation qui accompagne l’instrument un point d’exclamation dans un triangle équilatéral a pour but d’attirer l’attention de l’utilisateur sur des
instructions importantes de fonctionnment ou de maintenance. 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 ndirectement 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
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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 signo de admiración en un triángulo equilátero en el manual, advierte al usuario
sobre la presencia de instrucciones importantes de operación y mantenimiento del aparato. 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 specificaciones 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 publi
Wichtige Benutzerinformationen
Deutsch
Für ein vollständiges Verständnis und eine sichere Handhabung dieses Produktes ist es
notwendig, daß der Benutzer dieses Handbuch vollständig durchliest. Ein Ausrufezeichen in
einem gleichseitigen Dreieck soll den Benutzer auf die Anwesenheit wichtiger Betriebs und
Wartungsanweisungen in der dem Gerät beiliegenden Dokumentation 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
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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 Cup Loading Manifold • safety warnings and precautions
Informazioni importanti per l’operatore
Per un utilizzo sicuro del prodotto, leggere attentamente l’intero contenuto del presente
manuale. Il punto esclamativo all’interno di un triangolo equilatero indica all’operatore la
presenza di importanti istruzioni di funzionamento e manutenzione nella documentazione
allegata al prodotto. 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 Cup Loading Manifold
Unpacking
Unwrap all packages carefully and compare contents with the packing
list, making sure all items arrived. If any part(s) 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(s) appear 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.
Ettan IPGphor Cup Loading Manifold
Fig 1. Components of the Ettan IPGphor Cup
Loading Manifold system. Only one of each
item shown.
A.
B.
C.
D.
E.
F.
Manifold ceramic tray
Pre-cut electrode wicks
Lid adapter
Electrode assembly
Sample cups
Cleaning brush
G.
H.
I.
J.
K.
Pre-cut paper bridges
Sample cup insertion tool
Cleaning solution
Forceps
Spirit level
The Ettan™ IPGphor™ 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 and Ettan IPGphor II
Isoelectric Focusing Units. 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.
B.
E.
H.
A.
D.
G.
J.
C.
F.
I.
K.
• p1
Ettan IPGphor Cup Loading Manifold • Ettan IPGphor Cup Loading Manifold
Manifold
forcep wells
channel
numbering
centering
protrusion
The manifold (Fig 2) is made of aluminum oxide ceramic for efficient
heat transfer and temperature control during IEF. Protrusions along the
channel inside the manifold align the rehydrated IPG strip, keeping it
straight and centered when placed inside the manifold. The manifold has
a coating to minimize protein adsorption. Because some cleaning agents
can remove this coating, clean the manifold only with the Ettan IPGphor
Strip Holder Cleaner. The manifold is fragile and should be handled with
care. IPG strips from 7 cm to 24 cm long may be used. The unit may also
be used for IPG strip equilibration prior to second dimension electrophoresis. Forcep wells are located at the ends of the channels to aid strip
removal after electrophoresis.
Fig 2. Ceramic manifold with details.
Electrode Assembly
cam in open
position
hold down
teeth
electrode
teeth
cam in closed
position
Fig 3. Electrode and details.
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• p2
The movable electrodes (Fig 3) can be placed anywhere along the manifold where the electrode pins will make electrical contact with the power
supply pads on the bed of the Ettan IPGphor. The electrodes have a platinum wire that is strung across the bottom of the electrode teeth to
provide electrical contact to the IPG strips. The hold down teeth are
found along the opposite length of the electrode. They are used to apply
pressure on the paper bridges when the manifold is used for bridge loading. Cams on the sides of the electrode secure the electrodes in place and
must be in the closed position during IEF.
• Ettan IPGphor Cup Loading Manifold
Sample Cups
cup arms
cup feet
Fig 4. Sample loading cups.
Sample cups (Fig 4) can be placed almost anywhere along the length of
the Ettan IPGphor manifold that is not blocked by a centering protrusion. The sample cups are supplied in strips of 6 for easy handling and
placement. If fewer than 6 cups are required, they may be easily separated by cutting the thin plastic bridging the strip of cups together. The
sample cups can accommodate sample volumes of up to 150 µl. For
proper sealing of the cup to the gel, all of the feet of the sample cup must
rest on the bottom of the channel and all cup arms must be fully pressed
down into the channel.
Sample cup insertion tool
The sample cup insertion tool (Fig 5) is supplied with the manifold kit
and refill packages of sample cups. It is used for proper placement and
sealing of the cup over the IPG strips.
IEF electrode paper wicks
The small, precut rectangular wicks (Fig 6) must be placed at both the
anodal and cathodal ends of the rehydrated IPG strips just under the electrodes. The wicks absorb excess water, salts, and proteins with pIs that
lie outside the pH range of the IPG strip.
Fig 5. Sample cup insertion tool.
Fig 6. Paper wicks.
• p3
Ettan IPGphor Cup Loading Manifold • Ettan IPGphor Cup Loading Manifold
Paper bridge loading pads
Bridge loading pads (Fig 7) are used to load larger volumes of sample
(375–500 µl) than what can be held in a sample cup. The bridges are
0.8 cm × 5.0 cm in size with pointed ends. One end of the pad contacts
the IPG strip and is held in place by the hold down teeth of the electrode
assembly. The other end of the bridge loading pad makes contact with a
paper wick placed under the platinum wire of the electrode assembly.
Lid adaptor for standard strip holders
Fig 7. Paper bridges.
When using the IPGphor standard strip holders or the IPGphor cup loading strip holders a foam pad lid adapter (Fig 8) must be used to apply the
correct amount of pressure to the tops of the IPGphor strip holders and
keep them in contact with the IPGphor power supply pads. If running
only one IPGphor strip holder a second IPGphor strip holder must be
placed on the opposite side of the lid adapter to maintain the adapter in
a level position.
Not included but required:
Immobiline DryStrip Cover Fluid (order separately)
Fig 8. Standard strip holder adapter.
Immobiline DryStrip Cover Fluid is required to ensure that the rehydrated IPG strip gels do not dry out during electrophoresis. Without
cover fluid, the strips will dry out, urea will crystallize, and the sample
will not focus properly.
Immobiline DryStrip Reswelling Tray (order separately)
The Immobiline DryStrip Reswelling Tray is required for proper strip
rehydration. The channel in the Cup Loading Manifold is too wide to
ensure proper absorption of the required volumes of rehydration solution.
Two trays are available. One for 7–18 cm strip lengths and a second for
7–24 cm strip lengths.
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• p4
• IPGphor lid modifications
IPGphor lid modifications
The IPGphor lid must be modified before using the manifold system. The
three (3) foam pads on the lid must be removed (Fig 9). Once removed,
any adhesive remaining on the lid must be cleaned off. The foam pads are
NOT replaced on the lid. After lid modification, the lid adaptor (Fig 8)
must be used when running IPGphor strip holders.
foam pads in place
Fig 9. Lid modifications.
foam pads removed
• p5
Ettan IPGphor Cup Loading Manifold • instructions
Instructions
1
Rehydrate the IPG strips with the gel side down in the appropriate volume of
rehydration solution using the Immobiline DryStrip Reswelling Tray. Rehydration
in the Cup Loading Manifold is not recommended: the channel is too wide to
ensure proper rehydration. Follow the instructions included with the Immobiline
DryStrips. The table below is provided as a reference.
IPG strip
length (cm)
Rehydration
volume (µl)
7
125
11
200
13
250
18
340
24
450
2
Cover the IPG strips with Immobiline DryStrip Cover Fluid and allow the strips to
rehydrate overnight (10–20 hours).
3
Fig 10. Manifold placement on the Ettan IPGphor.
Important! Before proceeding, make sure the
Ettan IPGphor unit is placed on a level
surface.
Important! Ensure that the three foam pads
have been removed from the lid of the
Ettan IPGphor.
um 80-6499-52
• p6
Clean and dry the IPGphor bed before placing the manifold tray on the unit. Position
the manifold on the IPGphor platform. The small T-shaped protrusion fits into a cutout section of the IPGphor bed near the lid hinge (Fig 10). Ensure that the manifold
is level by placing the round spirit level on the center of the manifold tray after it is
placed on the Ettan IPGphor unit.
• instructions
4
Measure out 108 ml of Immobiline DryStrip Cover Fluid (even if fewer than
12 strips will be loaded into the manifold). Add the cover fluid evenly in the
12 manifold channels. Transfer the strips to the Ettan IPGphor Cup Loading
Manifold. Place the strips under the cover fluid face up in the tray with the
anodic (+, pointed) end of the IPG strip resting on the appropriate mark etched
into the bottom of the manifold channel (the end of the gel, not the end of the
plastic, should align with the etched mark). Center the strip down the length of
the manifold channel. Protrusions along the sides guide the strip approximately
straight, although some manual adjustment of the strip may be necessary (Fig 11).
Fig 11. Placement of IPG strips in manifold channels.
24 cm
18 cm
13 cm
11 cm
Note: If cathodic cup loading is going to be
used, the strips should be placed such that
the anodic end of the strips is 3–4 cm
beyond the etched placement mark.
7 cm
• p7
Ettan IPGphor Cup Loading Manifold • instructions
5
Place a strip of cups in the appropriate position (Fig 12), for example ~1 cm from
the end of the gel portion of the IPG strip. Do NOT place the cup with the feet
over a center protrusion. Push the cups into the channels with gloved fingers,
starting at one end of the strip and working towards the other. Align the insertion
tool over the cups and push down to ensure that the feet of the cups are properly
seated at the bottom of the channel (wiggle the tool gently while pushing down in
order to ensure that the cups are seated as far down as they will go). Take care not
to move the cups while removing the insertion tool. Fill the cups with cover fluid
to test for proper seating of the cups. Remove the cover fluid after 10 minutes.
incorrect cup position
Fig 12. Sample cup positioning details.
Note: Cups must not straddle the
centering protrusions on the
bottom of the channels.
correct cup position
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• p8
• instructions
6
Count out the appropriate number of precut paper wicks. Two wicks per strip are
required. Separate the wicks from each other. Add 150 µl distilled water to each
wick. Place the wicks on the IPG strips such that one end of the wick overlaps
the end of the gel on the IPG strip (Fig 13). The electrode must contact the wick.
With the electrode cams in the open position, place the electrode assembly on
top of all the wicks. Swivel the cams into the closed position under the external
lip of the tray. The electrodes should not be moved while the cams are in the
closed position (Fig 14).
7
Fig 13. Correct placement of paper wicks.
Briefly centrifuge the protein sample prior to loading to remove insoluble material
and particulate matter. These materials will impede sample entry and result in
vertical streaks in the second-dimension gel. Load samples into the sample cups.
A maximum of 150 µl of sample may be placed in these cups. Check to make
sure that there is cover fluid over the samples. When the cups are initially placed
on the manifold, cover fluid will flow into the cups as they are seated. When
sample is introduced into the cups, the sample will sink to the bottom of the cup
and contact the IPG strip.
Note: For basic IPG strips, superior focusing patterns are generally obtained when
the sample cup is placed as close to the anodic (+) electrode as possible.
8
Close the Ettan IPGphor lid. Program the Ettan IPGphor with the desired run
parameters. Ramping the voltage slowly while the sample is entering the IPG strip
will improve results. Optimal ramp, voltages and times, or Vhrs (volt-hours) totals
must be determined empirically for each sample type. Focusing after sample cup
application frequently requires fewer Vhrs than in-gel sample rehydration loading
methods, particularly on basic pH-range strips.
Fig 14. Placement of electrode on paper wicks.
Cams are in the open position.
Important! The Ettan IPGphor unit is capable of producing thousands of volts.
Before operating the unit, read and fully understand the Ettan IPGphor operating
instructions and warnings.
Fig 15. Placement of paper wick overlapping
end of gel, electrode on paper wick, and
sample cup on gel.
• p9
Ettan IPGphor Cup Loading Manifold • suggested run conditions
Suggested run conditions
Table 1. Guidelines for Ettan IPGphor protocols with Cup Loading Manifold for broad, medium, and narrow pH range
Immobiline DryStrip gels. Running conditions: Temperature 20 °C, Current 50 µA/strip.
Immobiline DryStrip gels
Running conditions for application method
Cup Loading
Length
(cm)
pH range(s)
Voltage mode
7
3–10
3–10 NL
4–7
6–11
11
13
18
24
Bridge Loading
Voltage
(V)
Duration
(h:min)
Volt-hours
(kVh)
Duration
(h:min)
Volt-hours
(kVh)
1 Gradient
2 Gradient
3 Step and Hold
Total
500
4000
5000
0:01
1:30
0:45
2:15
0.01
3.4
3.7
7.1
0:01
2:30
0:30
3:00
0.01
5.6
2.5
8.0
3–10
4–7
1 Gradient
2 Gradient
3 Step and Hold
Total
500
4000
8000
0:01
1:30
1:30
3:00
0.01
3.4
10.6
14.0
0:01
2:30
1:40
4:10
0.01
5.6
12.0
17.6
6–11
1 Gradient
2 Gradient
3 Step and Hold
Total
500
4000
8000
0:01
1:30
1:15
2:45
0.01
3.4
8.5
11.9
0:01
2:30
1:30
4:00
0.01
5.6
10.0
15.6
3–10
3–10 NL
4–7
1 Gradient
2 Gradient
3 Step and Hold
Total
500
4000
8000
0:01
1:30
1:50
3:50
0.01
3.4
13.5
16.9
0:01
2:30
2:10
4:40
0.01
5.6
15.2
20.8
6–11
1 Gradient
2 Gradient
3 Step and Hold
Total
500
4000
8000
0:01
1:30
1:40
3:10
0.01
3.4
11.6
15.0
0:01
2:30
1:50
4:20
0.01
5.6
13.4
19.0
3–10
3–10 NL
4–7
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
1:10
13:10
0.9
3.9
13.5
9.7
28.0
3:00
6:00
3:00
1:50
13:50
0.9
3.9
13.5
14.7
33.0
6–9
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
3:20
15:20
0.9
3.9
13.5
26.7
45.0
3:00
6:00
3:00
4:00
16:00
0.9
3.9
13.5
31.7
50.0
6–11
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
500
1000
8000
8000
1:00
2:00
3:00
1:05
7:05
0.5
1.5
13.5
8.5
24.0
3:00
2:00
3:00
1:30
9:30
1.5
1.5
13.5
11.5
28.0
Narrow
intervals§
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
5:10
17:20
0.9
3.9
13.5
41.7
60.0
3:00
6:00
3:00
6:00
18:00
0.9
3.9
13.5
47.7
66.0
3–10
3–10 NL
4–7
3–7
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
4:40
16:40
0.9
3.9
13.5
36.7
55.0
3:00
6:00
3:00
5:50
17:50
0.9
3.9
13.5
46.3
65.0
6–9
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
5:10
17:10
0.9
3.9
13.5
41.7
60.0
3:00
6:00
3:00
6:40
18:40
0.9
3.9
13.5
53.7
72.0
Narrow
intervals§
1 Step and Hold
2 Gradient
3 Gradient
4 Step and Hold
Total
300
1000
8000
8000
3:00
6:00
3:00
9:40
21:40
0.9
3.9
13.5
77.7
96.0
3:00
6:00
3:00
11:40
23:40
0.9
3.9
13.5
93.7
112.0
§ Narrow intervals = 3.5– 4.5, 4.0–5.0, 4.5–5.5, 5.0–6.0, and 5.5– 6.7.
um 80-6499-52
• p10
• troubleshooting
Troubleshooting first-dimension IEF:
Ettan IPGphor Isoelectric Focusing System employing the Cup Loading Manifold.
symptom
possible cause
remedy
Current is too low or zero
Electrical continuity is impeded.
Check the external electrode contacts: The electrodes at the bottom of the strip holder
(one at each end) must make metal-to-metal contact with the appropriate electrode
contact area.
Check the internal electrode contacts: The gel (which becomes visible because of the
dye in the rehydration solution) must contact both electrodes in the manifold through
the paper wicks and/or paper bridge parts.
Check that the IPG strip is fully rehydrated along its entire length. Electrical contact
at the electrodes is reduced by incomplete rehydration.
Check that the paper wicks are present and properly positioned.
Voltage too low or does
not reach maximum set value
Ettan IPGphor protocol settings
are incorrect for the experiment.
Check that the current limit is properly set.
Check that the actual number of Immobiline DryStrip gels on the Ettan IPGphor
platform is the same as the number of gels entered in the protocol.
Conductivity/ionic strength
is too high.
Prepare the sample to yield a salt concentration less than 10 mM. The recommended
IPG Buffer concentration is 0.5%. A maximum of 2% is advisable only if sample solubility is a problem. High conductivity can also arise from the use of poor quality urea
or other denaturants. Urea is also prone to decompose to charged breakdown products.
Higher conductivity salts and ionic impurities in the sample can raise the conductivity
of the strip.
Shorter length IPG strips (e.g. 7 cm strips) will not reach 8000 V. The distance
between the electrodes is shorter so that the voltage gradient (V/cm) required to reach
the 50 µA current limit is reached at a lower overall voltage.
Sample leaks from cup
Incorrect cup placement.
Check that the feet of the cups are resting on the bottom of the manifold channel.
Check for correct positioning of sample cup arms.
Check that the feet of the cups are not resting on a centering protrusion in the channel.
Incorrect strip placement.
Check that the strip is centered inside of the channel.
Current limit setting is too high.
Do not exceed the maximum recommended setting of 50 µA per Immobiline DryStrip gel.
Immobiline DryStrip gel is not
fully rehydrated.
Ensure that the Immobiline DryStrip gels are rehydrated with a sufficient volume of
rehydration solution. Remove any large bubbles trapped under the Immobiline DryStrip
gel after placing it on rehydration solution.
Immobiline DryStrip gels
dried out during IEF.
Always apply Immobiline DryStrip Cover Fluid to prevent dehydration of rehydrated
Immobiline DryStrip gels.
Immobiline DryStrips turn
white and opaque
after focusing
Immobiline DryStrip
gels dried out during IEF.
Always apply recommended amount of Immobiline DryStrip Cover Fluid to prevent
dehydration of rehydrated Immobiline DryStrip gels.
Immobiline DryStrip Cover
Fluid overflows from
Cup Loading Manifold
Excess cover fluid added.
Do not add more than the recommended volume. Ensure that the outside rim of the
tray does not have any oil on it.
Sparking or burning in
the Immobiline DryStrip gels
• p11
Ettan IPGphor Cup Loading Manifold • paper-bridge loading
Paper-bridge loading
Note: The application point (anodic or
cathodic) is an important factor for obtaining
good results.
Large sample volumes and large protein amounts can be applied using
paper bridge loading. A paper pad (paper bridge) is soaked with sample
and placed between the anodic end of the Immobiline DryStrip gel and
the electrode (375–500 µl sample can be applied using the paper bridge
pads supplied with the manifold). Solutions containing up to 5 mg
protein have been loaded on a 18 cm-long narrow pH range Immobiline
DryStrip gel. The rehydrated Immobiline DryStrip gel is first positioned
in the bottom of the manifold channel. Then the paper bridge with
sample is positioned, followed by a paper wick (Fig 16). With anodic
application the anode electrode is positioned as far out as possible in the
electrode assembly, while the cathode electrode is positioned close to the
end of the Immobiline DryStrip gel to ensure good contact between the
paper wick and Immobiline DryStrip gel.
electrode assembly
Fig 16. Electrode positioning (anodic end) on the
paper bridges.
sample cup
paper
wick
IPG strip
anode
Note: A single paper bridge can be used
with the 24 cm gel strip. A paper bridge
can be used on both ends of all other
strips at one time.
um 80-6499-52
• p12
paper bridge
hold down teeth
• care and cleaning
Care and cleaning
Ceramic Manifold
Important! Do not use strong acids, bases,
ketones, alcohols, or other reagents to clean
the covers, sample cups, or electrodes or the
parts may be damaged. Cups may be briefly
rinsed with ethanol if desired.
Gently clean the manifold after each use: Wash the manifold channels
with the Ettan IPGphor Strip Holder Cleaner. Rinse thoroughly with
water and then deionized water and allow the manifold to air dry. Do
not use abrasive cleaning agents or pads. Take care to avoid chipping
the manifold.
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.
Electrodes
Wash the electrode assemblies with the Ettan IPGphor Strip Holder
Cleaner. Rinse thoroughly with water then deionized water and allow
to air dry.
Customer service information
Technical service and repair
Amersham Biosciences offers complete technical support for all our
products. If you have any questions about how to use this product,
please call or fax your local Amersham Biosciences representative.
Ordering information
Replacement parts
Code number
Ettan IPGphor Cup Loading Manifold
80-6498-57
Sample cups, pack of 20 (6x)
80-6498-95
Paper electrode wicks, pack of 40 (6x)
80-6499-14
Paperbridge pads, pack of 20 (6x)
80-6499-33
Electrode set
80-6498-76
Lid adapter
80-6499-71
Cleaning brush
80-6505-98
Spirit level
80-6194-19
Forceps SS
80-6506-17
Accessory products
Immobiline DryStrip Reswelling Tray, 7–18 cm
1
80-6371-84
Immobiline DryStrip Reswelling Tray, 7–24 cm
1
80-6465-32
Immobiline DryStrip Cover Fluid
Ettan IPGphor Strip Holder Cleaner
1 liter
17-1335-01
950 ml
80-6452-78
• p13
Ettan, Immobiline, IPGphor, and PlusOne are trademarks of Amersham Biosciences Limited.
Amersham and Amersham Biosciences are trademarks
of Amersham plc.
© 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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