cytiva Ettan IPGphor Cup Loading Manifold User manual

Ettan IPGphor Cup Loading
Manifold
User Manual
cytiva.com

Table of Contents
1Introduction ........................................................................................................ 3
1.1 Important user information ....................................................................................................................... 4
2 Description .......................................................................................................... 6
2.1 Overview ............................................................................................................................................................ 7
2.2 Accessories ...................................................................................................................................................... 12
3 Operation ............................................................................................................ 13
3.1 Instructions ...................................................................................................................................................... 14
3.2 Run conditions ................................................................................................................................................ 21
4 Maintenance ....................................................................................................... 22
5 Troubleshooting ................................................................................................. 24
6 Reference information ....................................................................................... 27
Table of Contents
2 Ettan IPGphor User Manual 11003460 AE

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 4
1 Introduction
Ettan IPGphor User Manual 11003460 AE 3

1.1 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 documenta-
tion. 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 Cytiva'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.
Definitions
This user documentation contains safety notices (WARNING, CAUTION, and NOTICE)
concerning the safe use of the product. See definitions below.
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.
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.
1 Introduction
1.1 Important user information
4 Ettan IPGphor User Manual 11003460 AE

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 proce-
dures.
Typographical conventions
Software items are identified in the text by bold italic text.
Hardware items are identified in the text by bold text.
In electronic format, references in italics are clickable hyperlinks.
1 Introduction
1.1 Important user information
Ettan IPGphor User Manual 11003460 AE 5

2 Description
About this chapter
This section provides an overview of the Ettan IPGphor Cup Loading Manifold unit.
In this chapter
Section See page
2.1 Overview 7
2.2 Accessories 12
2 Description
6 Ettan IPGphor User Manual 11003460 AE

2.1 Overview
Introduction
The Ettan IPGphor Cup Loading Manifold, in combination with Cytiva'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.
Illustration of the Ettan IPGphor Cup
Loading Manifold
The illustration below shows the main components of the Ettan IPGphor Cup Loading
Manifold:
1
2
3
5
6
8
9
4
7
10
2 Description
2.1 Overview
Ettan IPGphor User Manual 11003460 AE 7

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
Manifold
Ceramic Manifold
The manifold (see following figure) 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 (acrylonitrile-
butadiene-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 23
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.
2 Description
2.1 Overview
8 Ettan IPGphor User Manual 11003460 AE

forcep wells
channel
numbering centering
protrusion
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
2 Description
2.1 Overview
Ettan IPGphor User Manual 11003460 AE 9

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 feet
cup arms
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.
2 Description
2.1 Overview
10 Ettan IPGphor User Manual 11003460 AE

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.
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 elec-
trode 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 Description
2.1 Overview
Ettan IPGphor User Manual 11003460 AE 11

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.
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 to 18 cm
strip lengths and a second for 7 to 24 cm strip lengths.
2 Description
2.2 Accessories
12 Ettan IPGphor User Manual 11003460 AE

3 Operation
About this chapter
This section provides instructions on how to operate the Ettan IPGphor Cup Loading
Manifold unit.
In this chapter
Section See page
3.1 Instructions 14
3.2 Run conditions 21
3 Operation
Ettan IPGphor User Manual 11003460 AE 13

3.1 Instructions
Rehydrating the IPG strips
Follow the instructions below to rehydrate the IPG strips.
Step Action
1Rehydrate 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.
2Follow 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
3Cover 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
1Clean and dry the IPGphor bed before placing the manifold on the unit.
Position the manifold on the IPGphor platform.
3 Operation
3.1 Instructions
14 Ettan IPGphor User Manual 11003460 AE

Step Action
2The 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
1Measure 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.
2Transfer 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 elec-
trode assembly accommodates at each end of the strip without hiding the
strip, see following figure, to be able to see the brome phenol blue moving
away at start.
3 Operation
3.1 Instructions
Ettan IPGphor User Manual 11003460 AE 15

Step Action
3Center 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
7 cm
11 cm
13 cm
18 cm
Strip length
(cm) 13 18 24
711
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.
Placing sample cups
Place the sample cups following the instructions below.
3 Operation
3.1 Instructions
16 Ettan IPGphor User Manual 11003460 AE

Step Action
1Place 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.
2Align 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.
3Fill 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 chan-
nels.
Placing paper wicks
Follow the instructions below to place the paper wicks.
Step Action
1Count 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.
3 Operation
3.1 Instructions
Ettan IPGphor User Manual 11003460 AE 17

Step Action
2Place 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.
3With 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
Paper wick
Cup
IPG Strip
3 Operation
3.1 Instructions
18 Ettan IPGphor User Manual 11003460 AE

Sample application (loading)
Follow the instructions below to load the sample.
Step Action
1Briefly 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.
2Load 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.
3Close 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 posi-
tioned in the bottom of the manifold channel. Then the paper bridge with sample is
positioned, followed by a paper wick (see following figure).
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 Immobi-
line DryStrip gel.
Note: The anodic application point is an important factor for obtaining good
results.
3 Operation
3.1 Instructions
Ettan IPGphor User Manual 11003460 AE 19

anode paper
bridge
hold down
teeth
paper
wick IPG strip
electrode assembly
3 Operation
3.1 Instructions
20 Ettan IPGphor User Manual 11003460 AE