Ettan™ IPGphor™ 3 Isoelectric Focusing Unit

Table. Guidelines for running 7–24-cm Immobiline DryStrip gels on Ettan IPGphor II Isoelectric Focusing Unit. Running conditions: Temperature 20 °C; current 50 μA per strip except where noted. See footnotes for information specific to the different strip lengths.

7-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.

11-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.
† To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.

13-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.
† To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.

18-cm strips

Note: When using IPGphor Manifold and 10 kV, set current limit to 75 μA per strip and follow step 1, 2, 3b and 4b. Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed voltage is 8000 V and current 50 μA per strip. Follow step: 1, 2, 3a, 4a.

* When using the IPGphor Cup Loading Strip Holder, running crude samples, or when a more convenient overnight run of 15–17 h is desired, the time in step 2 can be prolonged up to 8 h with a duration of 6.0 kVh. Using this option, step 4 can be reduced by the added kVh in step 2, to reach the specified total kVh.

24-cm strips

Note: When using IPGphor Manifold and 10 kV, set current limit to 75 μA per strip and follow step 1, 2, 3b and 4b. Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed voltage is 8000 V and current 50 μA per strip. Follow step: 1, 2, 3a, 4a.

* When using the IPGphor Cup Loading Strip Holder, running crude samples, or simply to adapt the run time to a convenient overnight run of 15–17 h, the time in step 2 can be prolonged by up to 8 h with a duration of 5.2 kVh. Using this option, step 4 can be reduced by the added kVh in step 2, to reach the specified total kVh.

Fig 1. Positioning the manifold.

1. Position the manifold on the Ettan IPGphor platform. The small T-shaped protrusion fits into a cutout section of the Ettan IPGphor platform near the lid hinge.

2. 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.

3. 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.

1. Select and prepare a rehydration solution .
Depending on sample and strip, select the appropriate rehydration solution. Three options are available:
• DeStreak Rehydration Solution with DeStreak reagent, urea, thiourea and CHAPS.
• 2× Sample buffer/rehydration solution stock 1, with urea, thiourea and CHAPS (recipe see Table 1).
• 2× Sample buffer/rehydration solution stock 2, with urea and CHAPS (recipe see Table 2).
DeStreak Rehydration Solution contains DeStreak reagent and optimized concentrations of urea, thiourea and CHAPS. DeStreak in the rehydration solution is recommended for strips with a pH above .

If sample is added in the rehydration solution (when performing In-Gel rehydration), DeStreak Rehydration Solution is not recommended. The pH is adjusted by addition of IPG Buffer with same pH interval as the IPG strip used.

Table 1. 2× Sample buffer/rehydration solution stock 1.

Small aliquots (e.g. 2.5 ml) can be stored at -20ºC. Stable for 6 months.

Table 2. 2× Sample buffer/rehydration solution stock 2.

Small aliquots (e.g. 2.5 ml) can be stored at -20ºC. Stable for 6 months.

2. Place the IPGbox on a leveled table and place a Reswell Tray in the IPGbox.

3. Apply rehydration solution, see Table 3.

Note: For complete sample uptake, do not apply excess rehydration solution.

Table 3. Rehydration solution volume per Immobiline DryStrip gel.

¹ Including sample, if applied.

4. Carefully pull off the cover film from Immobiline DryStrip gel and place Immobiline DryStrip gel into the slot, gel-side down. Distribute the rehydration solution evenly under the strip. Gently lift and lower the strip and slide it back and forth along the surface of the solution to get complete and even wetting of the entire gel.

Note: Ensure that no bubbles are trapped under the Immobiline DryStrip gel.


Fig 1. Example of IPG strips in a Reswell Tray.

5. Gently close the lid of the IPGbox and allow the Immobiline DryStrip gels to rehydrate at room temperature for 10-24 hours. Overnight is recommended.

6. Use the Immobiline DryStrip gels within 20 minutes of opening the IPGbox. Discard the used Reswell Tray.

Note: Do not store the Reswell Tray in IPGbox. Long time pressure of a Reswell Tray can reduce the life span of the IPGbox Insert.

1. A maximum of 150 μl of sample may be placed in these cups.

2. Check to make sure that there is cover fluid over the samples. When the sample is introduced into the cups, it will sink through the oil to the bottom of the cup and come into contact with the IPG strip.

3. Close the Ettan IPGphor lid.

4. Start the Ettan IPGphor from the Ettan IPGphor Control Software or Program and run the Ettan IPGphor from its own control panel display.

For detailed instructions on installation and usage of Ettan IPGphor Control Software, and also on computer instrument connection, please refer to the Ettan IPGphor Software User Manual.

For Creating, editing, and running protocol programs from the Ettan IPGphor control panel, please refer to the Ettan IPGphor 3 Instrument User Manual.

Fig 4. Load and cover samples.

When using the IPGphor standard strip holders, lid adapters (see Fig. 11) must be used to apply the correct amount of pressure to the lids of the IPGphor strip holders. The pressure keeps the IPG strip in contact with the IPG strip holder electrodes.


Fig 11. Lid adapter.

To ensure good electrical contact when using the fixed length strip holder:

1. Apply two lid adapters right across the lids of the strip holders, one over the anodic area and the other over the cathodic area, see Fig 12.


Fig 12. Safety lid and lid adapters.

2. If running only one strip holder, place a second empty strip holder on the opposite side of the platform to balance the lid adaptor correctly (see Fig 12.). Distribute multiple stripholders evenly across the platform.

3. Carefully close the safety lid by applying light downwards pressure, ensure the mechanism locks the lid.

To inspect the run the light protective cover (see figure below) can be lifted without stopping the run.



Fig 13. To open the safety lid.

Press down the lid and the lock is released and the run is stopped. Close the lid and the run continues.

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.

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.


Fig 1. Strip holder.

IMPORTANT! Handle the ceramic holders with care, as they are brittle and fragile.

1. Select the strip holder(s) corresponding to the IPG strip length chosen for the experiment

2. Wash each holder with the strip holder cleaning solution supplied to remove residual protein.

3. Rinse thoroughly with double distilled water.

4. 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.

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:
1. Set the rehydration time to 0:00 and program Step 1 of the protocol for low voltage (30 –100 V) for 10–12 h.
2. Program additional steps as desired to achieve complete focusing.

Fig 3. Remove protective film from Immobiline DryStrip gel, starting at the acid (+) end, to prevent damage of the basic end of the gel which is generally softer.


Fig 4. Position it with the gel.

1. Position it with the gel side down and the anodic (+) end of the strip directed toward the pointed end of the strip holder.

2. Anodic (+) 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.


Fig 5. Lay entire IPG strip in the strip holder.

3. The anodic (+) end of the strip should be positioned as close to the top (pointed) end of the strip holder as posible. The gel can be visually identified once the rehydration solution begins to dye the gel. Be careful not to trap bubbles under the strip.


Fig 6. Protein sample can be applied at sample application well following the rehydration step if the protein sample was not included in the rehydration solution.

Fig 7. Apply Immobiline DryStrip Cover Fluid to minimize evaporation and urea crystallization.

1. Pipet the cover fluid drop wise into one end of the strip holder until one half of the strip is covered.

2. Then pipet the cover fluid drop wise into the other end of the strip holder, adding fluid until the entire IPG strip is covered.


Fig 8. Position the cover on the holder.

Pressure blocks on the underside of the cover ensure that the strip maintains good contact with the electrodes as the gel rehydrates.

The Ettan IPGphor platform has two electrode areas:

• The larger area is the positive electrode (anode)

• 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. Guide marks along the sides of the platform show approximate positioning for each strip holder size (7, 11, 13, 18, and 24 cm).


Fig 10. Ettan IPGphor 3 electrode areas.

Table. Guidelines for running 7–24-cm Immobiline DryStrip gels on Ettan IPGphor II Isoelectric Focusing Unit. Running conditions: Temperature 20 °C; current 50 μA per strip except where noted. See footnotes for information specific to the different strip lengths.

7-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.

11-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.
† To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.

13-cm strips

* When running crude samples, step 1 may be extended up to 4 h to allow salt to migrate out of the strip at low voltage.
† To convert this to a convenient overnight run, extend Step 1 to 6 h (3 kVh) and reduce step 4 by 3 kVh.

18-cm strips

Note: When using IPGphor Manifold and 10 kV, set current limit to 75 μA per strip and follow step 1, 2, 3b and 4b. Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed voltage is 8000 V and current 50 μA per strip. Follow step: 1, 2, 3a, 4a.

* When using the IPGphor Cup Loading Strip Holder, running crude samples, or when a more convenient overnight run of 15–17 h is desired, the time in step 2 can be prolonged up to 8 h with a duration of 6.0 kVh. Using this option, step 4 can be reduced by the added kVh in step 2, to reach the specified total kVh.

24-cm strips

Note: When using IPGphor Manifold and 10 kV, set current limit to 75 μA per strip and follow step 1, 2, 3b and 4b. Using IPGphor Regular Strip Holder or Cup Loading Strip Holder with the 18- and 24-cm strips, the maximum allowed voltage is 8000 V and current 50 μA per strip. Follow step: 1, 2, 3a, 4a.

* When using the IPGphor Cup Loading Strip Holder, running crude samples, or simply to adapt the run time to a convenient overnight run of 15–17 h, the time in step 2 can be prolonged by up to 8 h with a duration of 5.2 kVh. Using this option, step 4 can be reduced by the added kVh in step 2, to reach the specified total kVh.

1. With the electrode cams in the open position, position the electrode assembly on top of all the wicks. The electrode must be in contact with the wick.

2. 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 3. Positioning electrode assembly.

Rehydration can proceed on the bench top or on the Ettan IPGphor 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 protocol. This is especially convenient if temperature control during rehydration is a concern, or if a low voltage is applied during rehydration.

Note: Active rehydration (20-120 V) can also be performed if sample is included. 

Recommended sample loads

It is usual to include your sample in rehydration buffer. Sample should be free of particulates and prepared to be free of buffer salts, and other ionic contaminants (e.g. use 2-D Clean-up kit)

Table 1. Suitable sample loads* for silver and Coomassie staining using cup loading and rehydration loading.

* When using cup loading, an increased sample concentration will lead to an increased risk of protein precipitation in the sample cup.The volume is limited to 100 ul in the sample cup and the sample concentration should not exceed 1 mg/ml.
† Immobiline DryStrip gels, pH intervals: 3–5.6 NL,5.3–6.5, 6.2–7.5, and 7–11 NL.
§ Immobiline DryStrip gels, pH intervals: 3–5.6 NL, 5.3–6.5, 6.2–7.5 and 7–11 NL.
§§ Immobiline DryStrip gels, pH intervals: 3–5.6 NL, 5.3–6.5, 6.2–7.5, 7–11 NL and 3.5–4.5.

Apply the rehydration solution


Fig 2. Apply appropriate volume rehydration solution to the Strip Holder.

For typical composition of rehydration solution see either Table 2 or Table 3. For the correct volume to each strip see Table 4.

Table 2. Urea rehydration stock solution.
(8 M urea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 ml)*

* DTT is added just prior to use: 7 mg DTT per 2.5-ml aliquot of rehydration stock solution. For rehydration loading, sample is also added
to the aliquot of rehydration solution just prior to use.
† If necessary, the concentration of urea can be increased to 9 M or 9.8 M.
‡ Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl
glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).
§ As an alternative to IPG Buffer, use Pharmalyte 3–10 for Immobiline DryStrip 3–10 or 3–10 NL, Pharmalyte 5–8 for Immobiline DryStrip 4–7.

Store in 2.5-ml aliquots at -20 °C.

Table 3. Thiourea rehydration stock solution.
(7 M urea,2 M thiourea, 2% CHAPS, 0.5/2% Pharmalyte or IPG Buffer, 0.002% bromophenol blue, 25 ml)*

* DTT is added just prior to use: 7 mg DTT per 2.5-ml aliquot of rehydration stock solution. For rehydration loading, sample is also added to the aliquot of rehydration solution just prior to use.
‡ Other neutral or zwitterionic detergents may be used at concentrations up to 2% (w/v). Examples include Triton X-100, NP-40, octyl glucoside, and the alkylamidosulfobetaine detergents ASB-14 and ASB-16 (Calbiochem).

Store in 2.5-ml aliquots at -20 °C.

Table 4. Rehydration solution volume per Immobiline DryStrip gel.

¹ Including sample, if applied.

1. Deliver the solution slowly at a central point in the strip holder channel away from the sample application wells.
2. Remove any larger air bubbles.

1. Transfer the strips to the Ettan IPGphor Cup Loading Manifold.


Fig 2. Ettan IPGphor Manifold is a accessory for Ettan IPGphor IEF unit allowing electrophoresis of large sample loads on IPG strip lengths from 7-24 cm.

2. Position the strips under the cover fluid face up in the tray with the anodic (+) end of the IPG strip pointing at the anode of the Ettan IPGphor.

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

Sample cups

1. Position a strip of sample cups in the appropriate position, 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.

2. Ensure that the feet of the cups are properly seated at the bottom of the channel. Use the insertion tool, wiggle the tool gently while pushing it down.

3. Fill the cups with cover fluid to test for proper seating of the cups.

4. Remove the cover fluid after 10 minutes.

Electrode paper wicks

1. Add 150 μl distilled water to each paper wick.

2. Position the wicks on each end of the IPG strips so that one end of the wick overlaps the end of the gel on the IPG strip.

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.


Fig 9. Applying sample after rehydration

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).