Ettan™ IPGphor™ Cup Loading Manifold

Ettan™ IPGphor™ Cup Loading Manifold
GE Healthcare
Life Sciences
Ettan™ IPGphor™
Cup Loading Manifold
User Manual
Table of Contents
Table of Contents
1
2
Introduction ..........................................................................................................
5
1.1
Important user information .............................................................................................................
5
Description ............................................................................................................
7
2.1
2.2
Overview ...................................................................................................................................................
Accessories ..............................................................................................................................................
7
13
Operation ..............................................................................................................
15
3.1
3.2
Instructions ..............................................................................................................................................
Run conditions .......................................................................................................................................
15
23
4
Maintenance .........................................................................................................
25
5
Troubleshooting ...................................................................................................
27
6
Reference information ........................................................................................
31
3
Ettan IPGphor User Manual 11-0034-60 AD
3
1 Introduction
1
Introduction
Purpose of the User Manual
The User Manual provides you with the instructions needed to use Ettan IPGphor Cup
Loading Manifold together with an Ettan IPGphor unit.
In this chapter
Section
See page
1.1 Important user information
1.1
5
Important user information
Read this before operating Ettan
IPGphor unit
All users must read the entire Ettan IPGphor Operating Instructions before installing,
operating or maintaining the instrument. Always keep the Operating Instructions at
hand when using Ettan IPGphor.
Do not operate Ettan IPGphor in any other way than described in the user documentation.
If you do, you may be exposed to hazards that can lead to personal injury and you may
cause damage to the equipment.
Intended use
The Ettan IPGphor Cup Loading Manifold in combination with GE Healthcare's Ettan
IPGphor, Ettan IPGphor II and Ettan IPGphor 3 Isoelectric Focusing Units, 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.
Ettan IPGphor is intended to be used as a laboratory device for research purposes.
Ettan IPGphor shall not be used in any clinical procedures, or for diagnostic purposes.
Ettan IPGphor User Manual 11-0034-60 AD
5
1 Introduction
1.1 Important user information
Safety notices
This user documentation contains WARNINGS, CAUTIONS and NOTICES concerning the
safe use of the product. See definitions below.
Warnings
WARNING
WARNING indicates a hazardous situation which, if not avoided,
could result in death or serious injury. It is important not to proceed
until all stated conditions are met and clearly understood.
Cautions
CAUTION
CAUTION indicates a hazardous situation which, if not avoided,
could result in minor or moderate injury. It is important not to proceed until all stated conditions are met and clearly understood.
Notices
NOTICE
NOTICE indicates instructions that must be followed to avoid
damage to the product or other equipment.
Notes and tips
Note:
A Note is used to indicate information that is important for trouble-free and
optimal use of the product.
Tip:
A tip contains useful information that can improve or optimize your procedures.
Typographical conventions
Software items are identified in the text by bold italic text. A colon separates menu levels,
thus File:Open refers to the Open command in the File menu. Hardware items are
identified in the text by bold text (e.g., Power switch).
6
Ettan IPGphor User Manual 11-0034-60 AD
2 Description
2
Description
About this chapter
This section provides an overview of the Ettan IPGphor Cup Loading Manifold unit.
In this chapter
This chapter contains the following sections:
Section
2.1 Overview
2.2 Accessories
2.1
See page
7
13
Overview
Introduction
The Ettan IPGphor Cup Loading Manifold, in combination with GE Healthcare's Ettan
IPGphor, Ettan IPGphor II and Ettan IPGphor 3 Isoelectric Focusing Units, 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. IEF is performed with
the gel side facing up.
Samples up to 150 μl are applied in 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). The manifold can accommodate anodic and cathodic cup loading.
Larger volumes of the sample, 375-500 μl, are applied with paper bridge application.
Sample volumes up to the volume of the strip (18 cm strip = 340 μl) can be applied by in
gel rehydration loading.
Ettan IPGphor User Manual 11-0034-60 AD
7
2 Description
2.1 Overview
Illustration of the Ettan IPGphor
Cup Loading Manifold
The illustration below shows the main components of the Ettan IPGphor Cup Loading
Manifold:
2
4
7
1
3
6
9
5
8
8
10
Part
Function
Part
Function
1
Manifold
6
Pre-cut paper bridges
2
Pre-cut electrode wicks
7
Sample cup insertion tool
3
Electrode assembly
8
Cleaning solution
4
Sample cups
9
Forceps
5
Cleaning brush
10
Spirit level
Ettan IPGphor User Manual 11-0034-60 AD
2 Description
2.1 Overview
Manifold
Ceramic Manifold
The manifold (see figure below) 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.
Note:
The manifold is fragile and should be handled with care.
Manifold Light
The manifold light is made of the polymers PC (polycarbonate) and ABS (acrylonitrilebutadiene-styrene).
The manifold light's flat design is important for the heat transfer. If exposed to very high
temperatures, the flatness could be affected. Refer to Manifold Light, on page 26 regarding
cleaning temperature.
General information
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.
forcep wells
channel
numbering
Ettan IPGphor User Manual 11-0034-60 AD
centering
protrusion
9
2 Description
2.1 Overview
Electrode assembly
The movable electrodes, as shown below, can be placed along the manifold to fit the
various length of Immobiline Dry Strip. Electrode pins 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.
cam in open
position
hold down
teeth
electrode
teeth
cam in closed
position
10
Ettan IPGphor User Manual 11-0034-60 AD
2 Description
2.1 Overview
Sample cups
Sample cups, as shown in the figure below, can be placed 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.
cup arms
cup feet
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11
2 Description
2.1 Overview
Sample cup insertion tool
The sample cup insertion tool, shown below, 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, shown below, 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.
12
Ettan IPGphor User Manual 11-0034-60 AD
2 Description
2.1 Overview
Paper bridge loading pads
Paper bridge loading pads, as shown below, are used to load large volumes of sample
(375–500 μl). The paper 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 paper bridge loading pad makes contact with
a paper wick placed under the platinum wire of the electrode assembly.
2.2
Accessories
Introduction
The following accessory items are not supplied with the unit and need to be ordered
separately.
Immobiline DryStrip Cover Fluid
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.
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13
2 Description
2.2 Accessories
Immobiline DryStrip Reswelling
Tray
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.
14
Ettan IPGphor User Manual 11-0034-60 AD
3 Operation
3
Operation
About this chapter
This section provides instructions on how to operate the Ettan IPGphor Cup Loading
Manifold unit.
In this chapter
This chapter contains the following sections:
Section
3.1
See page
3.1 Instructions
15
3.2 Run conditions
23
Instructions
Rehydrating the IPG strips
Follow the instructions below to rehydrate the IPG strips.
Step
Action
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.
Ettan IPGphor User Manual 11-0034-60 AD
15
3 Operation
3.1 Instructions
Step
Action
2
Follow the instructions included with the Immobiline DryStrips. The table
below is provided as a reference.
3
IPG strip length (cm)
Rehydration volume (μl)
7
125
11
200
13
250
18
340
24
450
Cover the IPG strips with Immobiline DryStrip Cover Fluid and allow the strips
to rehydrate overnight (10–20 hours).
Placing the Manifold on the IEF
unit
Follow the instructions below to place the manifold on the IEF unit.
Step
Action
1
Clean and dry the IPGphor bed before placing the manifold on the unit.
Position the manifold on the IPGphor platform.
16
Ettan IPGphor User Manual 11-0034-60 AD
3 Operation
3.1 Instructions
Step
Action
2
The small T-shaped protrusion fits into a cutout section of the IPGphor bed
near the lid hinge, see figure below. Ensure that the manifold is level by
placing the round spirit level on the center of the manifold after it is placed
on the Ettan IPGphor unit.
Transferring the IPG strips to the
Manifold
NOTICE
Make sure that the Ettan IPGphor unit is placed on a stable flat
surface.
Follow the steps below to transfer the IPG strips to the manifold.
Step
Action
1
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.
Ettan IPGphor User Manual 11-0034-60 AD
17
3 Operation
3.1 Instructions
Step
Action
2
Transfer the strips to the Ettan IPGphor Cup Loading Manifold. Place the
strips under the cover fluid with the gel surface face up and the anodic (+,
pointed) end to the anode of the Manifold. Place the strip so that the electrode
assembly accommodates at each end of the strip without hiding the strip,
see figure below, to be able to see the brome phenol blue moving away at
start.
3
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, as shown below.
24 cm
18 cm
13 cm
11 cm
7 cm
Strip length
(cm)
7
11
13
18
24
Note:
Using 24 cm long Immobiline Dry Strip and cathodic cup loading is going to
be used, the strips should be placed 3–4 cm further up compared to the figure
above to make the cathode electrode assembly accommodated at the cathode
end, not hiding the sample application place.
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Ettan IPGphor User Manual 11-0034-60 AD
3 Operation
3.1 Instructions
Placing sample cups
Place the sample cups following the instructions below.
Step
Action
1
Place a strip of cups in the appropriate position as shown in the figure below,
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.
2
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.
3
Fill the cups with cover fluid to test for proper seating of the cups. Remove
the cover fluid after 10 minutes.
incorrect cup position
correct cup position
Note:
Cups must not straddle the centering protrusions on the bottom of the channels.
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19
3 Operation
3.1 Instructions
Placing paper wicks
Follow the instructions below to place the paper wicks.
20
Step
Action
1
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.
2
Place the wicks on the IPG strips such that one end of the wick overlaps the
end of the gel on the IPG strip, as shown in figure below. The electrode must
contact the wick.
Ettan IPGphor User Manual 11-0034-60 AD
3 Operation
3.1 Instructions
Step
Action
3
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, see figure below.
Electrode
Cup
Paper wick
IPG Strip
Sample application (loading)
Follow the instructions below to load the sample.
Step
Action
1
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.
Ettan IPGphor User Manual 11-0034-60 AD
21
3 Operation
3.1 Instructions
Step
Action
2
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 at the anodic (+) end of the Immobiline Dry Strip.
3
Close the Ettan IPGphor lid.
Paper-bridge loading
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.
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 (see figure below).
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.
Note:
The anodic application point is an important factor for obtaining good results.
electrode assembly
paper
wick
anode
22
IPG strip
paper
bridge
hold down
teeth
Ettan IPGphor User Manual 11-0034-60 AD
3 Operation
3.2 Run conditions
3.2
Run conditions
Introduction
Program the Ettan IPGphor with the recommended run parameters. Ramping the voltage
slowly while the sample is entering the IPG strip will improve results.
Instructions for optimal ramp, voltages and times, or Vhrs (volt-hours) totals are attached
to each package of Immobiline Dry Strip.
WARNING
Ettan IPGphor is a high voltage instrument that can cause fatal
electrical shock if the safety features are disabled. The safety lid
must be securely closed before starting a protocol.
WARNING
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.
Ceramic manifold
Follow the recommended run conditions found in the Instructions attached to each Immobiline DryStrip package.
Manifold Light
Follow the recommended run conditions found in the Instructions attached to each Immobiline DryStrip package except for focusing time.
CAUTION
Do not use any organic solvents during cleaning.
Focusing time
As a guideline, use the lower number of recommended volt-hours to avoid overfocusing.
Normally the focusing results does not improve after this time.
Ettan IPGphor User Manual 11-0034-60 AD
23
3 Operation
3.2 Run conditions
WARNING
Check that the high voltage is turned off before opening the safety
lid. The HV ON indicator on the front panel should not light.
24
Ettan IPGphor User Manual 11-0034-60 AD
4 Maintenance
4
Maintenance
Precautions
WARNING
Disconnect power. Always disconnect power from the instrument
before performing any maintenance task.
NOTICE
The ceramic strip holders and manifold are very brittle and fragile.
Take care in handling and do not subject to impacts.
NOTICE
Avoid scratching the electrode surfaces.
NOTICE
Keep the instrument dry and clean. Wipe regularly with a soft damp
tissue and, if necessary, a mild cleaning agent. Let the instrument
dry completely before use.
Ceramic Manifold
NOTICE
The ceramic strip holders and manifold trays are very brittle and
fragile. Handle carefully: Do not bump or drop.
Ettan IPGphor User Manual 11-0034-60 AD
25
4 Maintenance
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.
Manifold Light
Clean the Ettan IPGphor Manifold Light with water, no warmer than 40 °C, and with IPG
Strip Holder cleaning solution.
NOTICE
Do not use any organic solvents.
Electrodes
Wash the electrode assemblies with the Ettan IPGphor Strip Holder Cleaner. Rinse thoroughly with water then deionized water and allow to air dry.
NOTICE
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.
26
Ettan IPGphor User Manual 11-0034-60 AD
5 Troubleshooting
5
Troubleshooting
Troubleshooting first-dimension
IEF
The following table specifies the troubleshooting situations that you might encounter
while using the 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, wet and
properly positioned.
Ettan IPGphor User Manual 11-0034-60 AD
27
5 Troubleshooting
Symptom
Possible cause
Remedy
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.
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. Salts
and ionic impurities in the
sample can raise the conductivity of the strip.
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.
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.
28
Ettan IPGphor User Manual 11-0034-60 AD
5 Troubleshooting
Symptom
Possible cause
Remedy
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.
Sparking or burning in the
Immobiline DryStrip gels
Immobiline DryStrips turn
white and opaque after
focusing
Ettan IPGphor User Manual 11-0034-60 AD
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 75 μA to 18 and 24
cm strips and 50 μA to 7,
11 and 13 cm strips 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 DryStrip gels
dried out during IEF.
Always apply recommended amount of Immobiline
DryStrip Cover Fluid to
prevent dehydration of
rehydrated Immobiline
DryStrip gels.
29
5 Troubleshooting
30
Symptom
Possible cause
Remedy
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.
Ettan IPGphor User Manual 11-0034-60 AD
6 Reference information
6
Reference information
Technical service and repair
GE Healthcare offers complete technical support for all our products. If you have any
questions about how to use this product, please contact your local GE Healthcare representative.
Ordering information
The following table provides you with information about replacement parts and their
code numbers.
Replacement part
Code number
Ettan IPGphor Cup Loading Manifold, Ceramic
80-6498-57
Ettan IPGphor Cup Loading Manifold Light
11-0025-80
Sample cups, pack of 20 (6x)
80-6498-95
Paper electrode wicks, pack of 40 (6x)
80-6499-14
Paper bridge pads, pack of 20 (6x)
80-6499-33
Electrode set
80-6498-76
Cleaning brush
80-6505-98
Spirit level
80-6194-19
Forceps SS
80-6506-17
The following table provides you information about accessory parts and code number.
Accessory part
Quantity
Code number
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
1 liter
17-1335-01
IPGphor Strip Holder Cleaning Solution
1 liter
29-0119-27
Ettan IPGphor User Manual 11-0034-60 AD
31
For local office contact information, visit
www.gelifesciences.com
GE Healthcare Bio-Sciences AB
Björkgatan 30
751 84 Uppsala
Sweden
www.gelifesciences.com/2DE
GE, imagination at work and GE monogram are trademarks of General Electric
Company.
Ettan, Immobiline, IPGphor, and PlusOne are trademarks of GE Healthcare
companies.
© 2012 General Electric Company – All rights reserved.
First published 2005
All goods and services are sold subject to the terms and conditions of sale of
the company within GE Healthcare which supplies them. A copy of these terms
and conditions is available on request. Contact your local GE Healthcare representative for the most current information.
GE Healthcare Europe GmbH
Munzinger Strasse 5, D-79111 Freiburg, Germany
GE Healthcare UK Limited
Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK
GE Healthcare Bio-Sciences Corp.
800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA
GE Healthcare Japan Corporation
Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan
imagination at work
11-0034-60 AD 04/2012
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