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ROTIPHORESE® PROfessional IEF (1Y9Y.1)
Safety Information:
When used correctly, these units pose no health risk. However, these units can deliver dangerous levels of
electricity and are to be operated only by qualified personnel following the guidelines laid out in this instruction
manual. Anyone intending to use this equipment should read the complete manual thoroughly. The unit must never
be used without the safety lid correctly in position. The unit should not be used if there is any sign of damage to the
external tank or lid.
These units comply with the following European directives:
2014/35/EU Low Voltage Directive and 2014/30/UE (official Title 2004/108/EC) EMC Electromagnetic Compatibility
By virtue of the following harmonised standards:
BS EN IEC 61010-1: 2010 Safety Testing of Lab Equipment
BS EN IEC 61326-1:2013 EMC Electro Magnetic Compatibility
ROHS DIRECTIVE 2011/65/EU
BS EN 50581:2012 Restriction of Hazardous Substances
Packing List:
• 1 Main Unit → Base and Lid
• 1 Cooling Plate
• 2 Cathode Electrodes
• 2 Anode Electrodes
• 1 Glass Electrode Frame
• 2 Power Cables
• 1 IPG Strip Focusing tray
• 1 IPG Strip Rehydration Tray
Please verify, that you received the unit completely and without any damage. Any faults or losses have to be
reported to ROTH immediately. ROTH can not accept responsibility for goods, that were sent back without
informing them.
Please take a look at the packing list and check whether all components and accessories are present
Please retain all packaging material until the warranty period has expired.
Specification:
This Isoelectric focusing unit has a warranty against manufacturing and material faults of 12 months from date of
customer receipt. This warranty does not cover defects occurring by accident or misuse or defects caused by
improper operation.
Capacity
1 to 12 IPG Strips
7 to 24 cm long
IEF gel
Up to 24 cm x 22 cm
Platform dimensions
25 cm x 23 cm
Operating Conditions
Maximum Voltage
3000 V
Maximum Current
300 mA
Power
300 W
Platform Temperature
7-30 °C
Dimensions
58 cm x 45 cm x 13 cm
Weight
8,5 kg
Environmental Operating Conditions
Maximum Altitude
2000 m
Temperature range
4 °C – 65 °C
Humidity
Up to 80 %
Not for Outdoor use
Instructions for use
This apparatus is rated POLLUTION DEGREE 2 in accordance with IEC 664.
POLLUTION DEGREE 2, states that: “Normally only non-conductive pollution occurs.
Occasionally, however, a temporary conductivity caused by condensation must be expected”.
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Care and Maintenance
Cleaning of Isoelectric focusing Units:
The unit should be thoroughly rinsed with warm water or distilled water to prevent build up of salts but care should
be taken not to damage the plate electrodes and vigorous cleaning is not necessary or advised. Air drying is
preferable before use.
Units are best cleaned using warm water and a mild concentration of soap or other mild detergent.
➔ Water at temperatures above 60 °C can cause damage to the unit and components.
➔ Compatible detergents include dishwashing liquid, Hexane and Aliphatic hydrocarbons.
Note: The unit should not be left in detergents for more than 30 minutes.
➔ The units should never come into contact with the cleaning agents like Acetone, Phenol, Chloroform,
Carbon tetrachloride, Methanol, Ethanol, Isopropyl alcohol, Alkalis. These will cause irreversible and
accumulative damage.
RNase Decontamination:
RNase Decontamination can be performed using the following protocol:Clean the unit with a mild detergent as described above. Then wash with 3% hydrogen peroxide (H2O2) for 10
minutes and finally rinse with 0.1% DEPC- (diethyl pyrocarbonate) treated distilled water (e.g. Art. No. T143.1)
(Caution: DEPC is a suspected carcinogen. Always take the necessary precautions when using.)
Operating the IEF system
Setting up the cooling plate:
The unit is provided with a cooling plate which acts a platform for running the IPG strips and prevents overheating
of the strips when attached to a recirculating chiller. The cooling plate consists of two sealing ports which when
removed will seal the cooling plate. These should be removed before connecting to the chiller by pressing down on
the metal button at the top of each port.
Connecting the chiller to the cooling plate:
Connect the tubing from the chiller to both port sealers. Insert the port sealers onto the ports; this will now render
these in the open position.
Note: The chiller must be set between 16-17 °C for running IPG Strips and between 2-4 °C for running the Precast
Gels for Isoelectric Focusing. Switch on the chiller at least 10 minutes prior to electrophoresis to allow the cooling
plate to reach the desired temperature.
Running IPG Strips
IPG Strip Sample Rehydration
1. Lay individual IPG strips within the rehydration channels in the Hydration tray
2. Incubate IPG strips overnight with buffer (8M Urea, 1% CHAPS, 13mM DTT and 0.5% SERVALYT™ 3-
10, corresponding to the pH gradient of the IPG strip) containing SERVA proteome markers. At least 5
and 50 μg of protein is sufficient for 7 and 18 cm IPG strips respectively.
3. Overlay each strip with silicone oil to prevent desiccation during the overnight incubation period.
Electrophoresis
1. Place the unit on the bench in a convenient position.
2. Insert the cooling plate in the base unit. Ensure it locates properly with the pipes protruding from the
cuts in the side wall. Set the desired temperature of the chiller and switch it on at least 10 minutes prior
to electrophoresis to allow the cooling plate to reach the desired temperature.
3. Connect each adjustable electrode to the gold plugs fixed within the tank. The electrodes are colour
coded for correct polarity orientation.
4. Place the IPG strip focusing tray directly onto the cooling plate.
5. Using a forceps place the IPG strips into the running tray with the gel side facing upwards. Then cover
the anode and cathode ends of the IPG strips with electrode wicks saturated with deionised water.
Cover the IPG strips with enough Silicone oil before starting the run.
6. Gently clip both the electrodes over the anodic and cathodic ends of the IPG strip respectively.
7. Screw the power cables into the lid before placing it on top of the gel tank. Then connect the power
cables to the power supply and the unit is ready for Isoelectric Focusing.
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Recommended Running conditions for IPG strips
Recommended
Running conditions
for IEF of 7cm
SERVA IPG strip
Voltage Step
1 2 3 4 5
6 end
Voltage (V)
150
300
600
1500
3000
330
Time (h)
0.5
0.5
0.5
0.5
2.5
<20
Volt-hours
75
150
300
750
7500
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Recommended
Running Conditions
for IEF of 18cm
SERVA IPG strips
Voltage Step
1 2 3 4 5 end of Run
Voltage (V)
300
600
1500
3000
330
Time (h)
1 1 1
12.5
<20
Volt-hours
300
600
1500
37500
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Installation and Running Precast or Hand-cast Gels:
The composition and running of IEF gels varies considerably depending on the range of pH required, the
application (native or denaturing) and the format of the gels. The following instructions are general instructions for
Isoelectric focusing applications where the gels are hand poured or precast in a horizontal format on a gel support
(not supplied). Please consult a suitable reference text for gel compositions, recommended temperatures, voltage
and run times.
1. Place the unit on the bench in a convenient position.
2. Insert the cooling plate in the base unit. Ensure it locates properly with the pipes protruding from the cuts in
the side wall. Set the desired temperature of the chiller and switch it on at least 10 minutes prior to
electrophoresis to allow the cooling plate to reach the desired temperature.
3. For running precast gels place the electrode frame (1Y47.1) directly above the cooling plate, ensuring that
the pins within the frame are aligned with the holes of the cooling plate.
4. Apply 1-5ml of Triton X100 non-ionic detergent (or Kerosene) to the surface of the cooling plate. This will
act as a heat transfer agent between the cooling plate and the gel, during the run. Place the gel on its
support onto the Cooling plate in the position required, making contact with the applied Triton X100 on the
lower edge. Slowly lower the gel so that the Triton spreads under the gel support plate, expelling any air
and ensuring a good contact to the cooling plate surface. Remove all excess Triton from the cooling plate.
5. Prepare two electrode strips, one cathode, one anode, by cutting strips from filter paper which are slightly
shorter than the gel width. Moisten the Electrode strips with the relevant electrode solutions and drain the
strips if required.
6. Place the electrode strips 2mm from the cathode and anode edges of the gel. The etched positions will
help in positioning. Ensure the electrode strips are slightly shorter than the gel on which they are applied.
This will prevent electrical contact along the edges of the gel. If using a precast IEF gel samples may be
loaded by pipettes into the wells of a sample applicator strips (not supplied) overlaid across the width of the
gel at its centre. If using a precast agarose gel simply load the samples in the wells created by the comb.
7. Apply the samples and proceed with steps 9 to 13 if pre-focusing is not required. If prefocusing is required
then proceed with steps 9 to 13 before loading samples and repeating these steps. Pre-focusing is
generally performed at lower voltage than the following separation step.
1 2 3 4 5
6
4
8. Using the markings on the electrode holder which correspond to the positions of the electrodes on the
cooling plate, align the electrodes in the correct position. This is
achieved by loosening the screws on the electrode and sliding them
within the channel on the plate holder and retightening the location
screws.
9. Place the electrode into the unit and lower carefully so that the
platinum wire of the electrodes makes contact with the filter paper
wicks on the gel.
10. Connect the electrodes to their respective sockets in the base unit.
Then place the safety lid and connect to a suitable power supply.
11. The unit is now ready to be switched on for the run.
End of Run:
1. Once the run is complete turn of the power supply and the chiller. The chiller should be disconnected by
pressing the metal button on the sealing ports. If these are not used, liquid from the chiller can leak from
the chiller and cooling plate.
2. Unscrew the power cables and remove the safety lid.
3. Then carefully remove the electrode wicks without damaging the strips.
4. The IPG strips are now ready for fixation and staining, or SDS-PAGE for 2D electrophoresis.
IPG Strip Fixation and Staining:
1. SERVA Blue R (35051) is recommended for protein staining according to the protocol laid out below.
2. Immerse the IPG strips in 100ml of fixing solution (40ml ethanol, 10ml glacial acetic acid and 50ml water),
agitating gently for 30 minutes on a rocking platform set at 50 to 100rpm.
3. Mix together stock solution 1 (0.2% SERVA Blue R, 90% ethanol (P075.1)) and stock solution II (20%
acetic acid) before adding the IPG strips and incubating for a further 20 minutes on the rocking platform.
4. Begin destaining the IPG strips by rinsing them in fixing solution for 30 seconds.
5. Mix 20ml of ethanol with 10ml of glacial acetic acid and 70ml of distilled water, while the IPG strips are still
in the fixing solution. Transfer the IPG strips to the destaining solution and incubate the IPG strips until
discrete protein bands are visible upon a clear background.
6. Rinse the IPG strips for 5 minutes in distilled water. Repeat. The IPG strips should now be ready for
visualisation.
Strip preparation for second dimension SDS-PAGE:
IPG strips are often used in the first dimension of 2D-electrophoresis, where proteins are initially resolved by their
isoelectric point and, then, by their molecular weight, in the second dimension, using SDS-PAGE. However, before
second dimension SDS-PAGE can take place the strips must be equilibrated after first-dimension IEF.
1. Transfer the IPG strips from the PROfessional IEF unit to a clean rehydration tray (1YA3.1). Using
tweezers or forceps gently lower each strip, gel side upwards, into the bottom of each channel within the
rehydration tray. Cover each 7 and 18cm strip respectively with 3 and 7ml of equilibration buffer I (50mM
Tris-HCl, pH 8.8; 6M Urea; 30% glycerol; 2% SDS; 0.01% bromophenol blue and 1% (w/v) DTT). Incubate
gently for 10 minutes on an agitating platform.
2. Carefully remove equilibration buffer I from rehydration tray, taking care to prevent the IPG strips from
falling out of the tray. Add the same volumes of equilibration buffer II (50mM Tris-HCl, pH 8.8; 6M Urea;
30% glycerol; 2% SDS; 0.01% bromophenol blue and 5% iodoacetamide) to each channel, as described in
step 1.
3. Incubate for 10 minutes.
4. Remove each IPG strip from the tray and dip it briefly in 1 x Laemmli buffer, before overlaying the strip with
forceps along the top of a pre-made acrylamide gel, ensuring that there are no air bubbles between the
strip and the gel. Please note that only the 1-mm thick edge of IPG strip should touch the edge of the gel
otherwise resolution will be compromised.
5. Cover the strip with melted 0.5% (w/v) agarose to keep it in position on top of the gel. Once the agarose
has set, the gel is then ready for second-dimension electrophoresis.
General maintenance post IEF:
1. After use, remove the electrodes from the glass electrode frame and rinse them carefully with distilled
water to prevent corrosion by the strong acidic and basic solutions. DO NOT submerge the socket
connector.
2. Rinse the buffer chambers with distilled water, again taking care not to submerge the socket connector. Dry
the chambers by using a vacuum line avoiding damage to the platinum electrodes.
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Recommended Isoelectric Focusing Products and Replacements parts
PROfessional IEF Cooling Platform
1YA0.1
PROfessional IEF Focusing Tray
1YA6.1
PROfessional IEF Rehydration Tray
1YA3.1
PROfessional IEF Electrode Frame
1YA7.1
PROfessional IEF Glass Platform
CP55.1
PROfessional IEF Anode Electrode
CP52.1
PROfessional IEF Cathode Electrode
CP53.1
PROfessional IEF Spring Anode Electrode
1YA5.1
PROfessional IEF Spring Cathode Electrode
1YA4.1
PROfessional IEF Focusing Tray Positive Electrode
1YA2.1
PROfessional IEF Focusing Tray Negative Electrode
1YA1.1
ROTIPHORESE® PROfessional IEF 1Y9Y.1
Carl Roth GmbH + Co. KG
Schoemperlenstraße 3-5 • 76185 Karlsruhe • P.O. Box 100121 • 76231 Karlsruhe
Phone: +49 (0) 721/ 5606-0 • Fax: +49 (0) 721/ 5606-149 • info@carlroth.com • www.carlroth.com lha 11/2022
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