4 First dimension: IEF in individual IPG strips

The standard protocol described here is valid for broad gradients in the pH range between 3 and 12 (e.g. IPG 4-7, 4-9, 6-10, 3-10, or 8-12, as well as IPG 4-12 (Görg et al., 1998), and for narrow IPGs in the acidic and neutral range (e.g. IPG 5-6). For narrow pH gradients above pH 9 (e.g. IPG 9-12 or 10-12), a modified protocol has been established which is described in detail elsewhere (Görg et al. 1997).

Immobiline DryPlate 4-7, Immobiline DryStrips 4-7, Immobiline DryStrips 3-10L, Immobiline DryStrips 3-10NL (Amersham Pharmacia Biotech)

Pharmalyte (pH range 3-10), IPG buffers, repel silane (Amersham Pharmacia-Biotech), Serdolit MB-1 mixed bed ion ecxhanger resin, silicone oil (Serva), CHAPS (Sigma), urea (Merck)

Rehydration solution (8 M urea, 0.5% CHAPS, 15 mM DTT and 0.2% ampholyte). To prepare 50 ml of the solution, dissolve 25.0 g of urea in deionized water and complete to 50 ml. Add 0.5 g of Serdolit MB-1, stir for 10 min and filter. To 48 ml of this solution add 0.25 g of CHAPS, 100 mg of DTT, and 0.25 ml IPG buffers (alternatively: Pharmalyte pH 3-10) and complete to 50 ml with deionized water. Rehydration solution should be prepared freshly the day you use it.

Prior to IEF, cut the dried IPG gels -or the ready-made Immobiline DryPlates- into individual 3-4 mm wide strips with the help of a paper cutter (Fig. 5). Alternatively, ready-cut IPG strips (Immobiline DryStrip) can also be used. Then the desired number of IPG gel strips is rehydrated to the original gel thickness (0.5 mm).The rehydrated strips are then placed on the cooling block of the electrofocusing chamber, or in the grooves of the strip aligner of the tray of the Pharmacia DryStrip Kit. Samples are applied into silicone frames or sample cups placed directly on the surface of the IPG gel strips. Alternatively, samples can be applied directly by in-gel rehydration using Amersham Pharmacia’s reswelling tray, or IPG strip holders of Amersham Pharmacia’s IPGphor.

For analytical purposes, typically 50-100 µg of protein are applied onto a single, 180 mm long IPG gel strip, whereas for micropreparative purposes up to several mg of protein can be applied in case that Pharmacia's Immobiline DryStrip Kit with sample cups is used. When IEF is performed using the DryStrip Kit, the IPG gel strips can be covered by a layer of silicone oil which is advantageous when running very basic pH gradients (e.g. IPG 10-12) (Görg et al. 1997) or narrow pH gradients with long focusing times for micropreparative IPG-Dalt (Bjellqvist et al. 1993).

1. To rehydrate the IPG gel strips to their original thickness (0.5 mm), take two layers of Parafilm (roll, 50 cm wide) and cut out a U-frame which has the same shape and size as the the glass plate's U-frame. Assemble the rehydration cassette in that way that a 0.7 mm thick U-frame (0.5 mm original thickness plus two layers of Parafilm, 0.1 mm each to compensate for the 0.2 mm wide GelBond PAGfilm on which the IPG gel is polymerized) is obtained. If larger sample volumes (> 100 µl) are to be applied, it is advantageous to use one layer of parafilm only. Clamp the cassette together and fill in the rehydration solution either from the bottom with a syringe or from the top with a pipette.

2. Take an appropriate number of dry IPG gel strips and pull off their protective covers. Lower them carefully, but without delay, into the rehydration cassette, gel side towards the glass plate bearing the U-frame (Fig. 6). Allow the strips to rehydrate overnight at room temperature. Make sure that the cassette does not leak.

3. After the IPG gel strips have been rehydrated, pour the rehydration solution out of the reswelling cassette, remove the clamps and open the cassette with the help of a spatula. Using clean forceps, rinse the rehydrated IPG gel strips with deionized water for a second and place them, gel side up, on a sheet of water saturated filter paper. Wet a second sheet of filter paper with deionized water, blot it to remove ecxess water and put it onto the surface of the IPG gel strips. Blot them gently for a few seconds to remove ecxess rehydration solution in order to prevent urea crystallization on the surface of the gel during focusing.

1. Cover the flat-bed cooling block with 2-3 ml of kerosene and place the IPG gel strips (up to 40!) side by side, 1-2 mm apart, on it (Fig. 7). The acidic end of the IPG gel strips must face towards the anode!

2. Cut two IEF electrode strips or paper strips prepared from 2 mm thick filter paper (e.g. MN 440, Macherey & Nagel, Germany) to a length corresponding to the width of all IPG gel strips lying on the cooling plate. Soak the electrode strips with deionized water (however not Milli-Q quality!) and remove ecxessive moisture by blotting with filter paper.

3. Place the IEF electrode strips on top of the aligned IPG gel strips at the cathodic and anodic ends.

4. When running basic IPGs (e.g. IPG 6-10 or IPG 10-12), put an extra paper strip soaked with 20 mM DTT onto the IPG gel surface near the cathodic electrode strip.

6. Pipette the samples (20 µl each, preferably dissolved in Lysis buffer) into the silicone frames. Protein concentration should not ecxeed 5-10 mg/ml. Otherwise, protein precipitation at the sample application area may occur. To avoid this, it is advantageous to dilute the sample with Lysis buffer and apply larger volumes instead (cf. section 4.3.1.3).

7. Position the electrodes and press them gently down on top of the IEF electrode strips.

8. Place the lid on the electrofocusing chamber, connect the cables to the power supply and start IEF. Running conditions depend on the pH gradient and the length of the IPG gel strips used. An appropriate time schedule for orientation is given in Table 4 (Görg 1993). For improved sample entry, voltage is limited to 150 V (30 min) and 300 V (60 min) at the beginning. Then continue with maximum settings of 3500 V to the steady state. Optimum focusing temperature is 20°C (Görg et al. 1991).

9. After IEF, those IPG gel strips which are not used immediately for second dimension run and/or are kept for further reference are stored between two sheets of plastic film at -78 °C up to several months (Fig. 8).

The application of higher sample volumes (> 20 µl) is simplified when the sample cups of the Immobiline DryStrip Kit are used (Fig. 9). A sample volume of 100 µl can be applied at a time, and it is possible to apply a total of up to 200-300 µl, portion by portion, onto a single IPG gel strip. Typical sample load is 60-100 µg protein/strip for silver stained 2-D patterns, whereas for preparative purposes up to 10 mg of protein from a cell lysate can be loaded onto a single IPG gel strip.

If very basic pH gradients (pH > 10) or narrow pH gradients (pH-range <1 unit) for micropreparative runs are used, the IPG strips ought to be protected by a layer of silicone oil. In case of broad pH gradients not ecxeeding pH 10 (e.g. IPG 4-7, 4-9 or 6-10), the DryStrip Kit can be used without silicone oil overlay.

1. Place the cooling plate into the Multiphor II Electrophoresis unit. Pipette 3-4 ml of kero-sene or silicone oil onto the cooling plate and position the Immobiline DryStrip tray on the cooling plate. Avoid trapping large air bubbles between the tray and the cooling plate.

2. Connect the electrode leads on the tray to the Multiphor II unit.

3. Pour about 10 ml of of silicone oil into the tray.

4. Place the Immobiline strip aligner into the tray on top of the oil.

5. Transfer the rehydrated IPG gel strips (gel-side up and acidic end towards the anode) into adjacent grooves of the aligner in the tray. Align the strips such that the anodic gel edges are lined up (Fig. 9).

6. Cut two IEF electrode strips or paper strips prepared from 2 mm thick filter paper (e.g. MN 440, Macherey & Nagel, Germany) to a length corresponding to the width of all IPG gel strips lying in the tray. Soak the electrode strips with deionized water, remove ecxessive moisture by blotting with filter paper and place the moistened IEF electrode strips on top of the aligned strips near the cathode and anode.

7. Position the electrodes and press them gently down on top of the IEF electrode strips.

8. Put the sample cups on the sample cup bar. Place the cups high enough on the bar to avoid touching the gel surface. Put the sample cup bar in position so that there is a distance of a few millimeters between the sample cups and the anode (or cathode, in case of cathodic sample application).

9. Move the sample cups into position, one sample cup above each IPG gel strip, and finally press down the sample cups to ensure good contact with each strip.

10. Once the sample cups are properly positioned, pour about 80 ml of silicone oil into the tray so that the IPG gel strips are completely covered. If the oil leaks into the sample cups, suck the oil out, re-adjust the sample cups and check for leakage again. Fill up each sample cup with a few drows of silicone oil.

Note: In case of IEF using pH gradients in the range between pH 4 and 9, the oil-step can be omitted!

11. Pipette the samples into the cups by underlaying. Watch again for leakage!

12. Close the lid of the Multiphor II electrophoresis chamber and start the run according to the parameters given in Table 4. For improved sample entry, voltage is limited to low voltages (150-300 V) for the first few hours. Then continue with maximum settings of 3500 V to the steady state. Optimum focusing temperature is 20°C (Görg et al. 1991).

13. If sample volumes ecxeeding 100 µl are to be applied, pipette in 100 µl, run IEF with limited voltage until the sample has migrated out of the cup. Then apply another 100 µl and repeat the procedure until the whole sample has been applied.

14. When the IEF run is completed, remove the electrodes, sample cup bar and IEF electrode strips from the tray. Use clean forceps and remove the IPG gel strips from the tray. Those IPG gel strips which are not used immediately for second dimension run and/or are kept for further reference are stored between two sheets of plastic film at -78°C up to several months (Fig. 8).

Especially for micropreparative IPG-Dalt, samples can be applied by in-gel rehydration, which works for the majority of proteins (Rabilloud et al. 1994, Sanchenz et al. 1997). However, it is recommended to check, whether the high-molecular mass proteins, very basic proteins, and membrane proteins, respectively, have entered the gel matrix properly.

1. Cut the dried IPG gels into 3 mm wide strips with the help of a paper cutter (Fig. 5). Alternatively, use ready-made IPG dry strips.

2. Solubilize proteins with sample solubilization buffer (9 M urea, 2% CHAPS, 0.8% Pharmalytes 3-10, 15 mM DTT) and dilute the extract (protein conc.: ≈10 mg/ml) with sample solubilization buffer (dilution: 1+1 for micropreparative runs, and 1+19 for analytical runs, respectively) to a final volume of 400-500 µl for 180 mm long IPG strips.

3. Pipette 350-400 µl of sample-containing rehydration solution into the grooves of the reswelling tray (Fig. 10). Peel off the protective cover sheets from the IPG strips and insert the IPG strips (gel side-down) into the grooves. Avoid trapping air-bubbles. Cover the IPG strips with 1 ml of silicone oil, close the lid and let the strips rehydrate overnight.

4. After the IPG gel strips have been rehydrated, rinse them with deionized water for a second and place them, gel side up, on a sheet of water saturated filter paper. Wet a second sheet of filter paper with deionized water, blot it slightly to remove ecxess water and put it onto the surface of the IPG gel strips. Blot them gently for a few seconds to remove ecxess rehydration solution in order to prevent urea crystallization on the surface of the gel during IEF.

5. Cover the flat-bed cooling block with 2-3 ml of kerosene and place the IPG gel strips, side by side and 1-2 mm apart, on it. The acidic end of the IPG gel strips must face towards the anode! If IEF is to be performed under a protective layer of silicone oil, use Pharmacia’s DryStrip kit instead ( however, without sample cups) (cf. section 4.3.1.3).

6. Cut two IEF electrode strips to a length corresponding to the width of all IPG gel strips (and spaces between them) lying on the cooling plate. Soak the electrode strips with deionized water and remove ecxessive moisture by blotting with filter paper.

8. Position the electrodes and press them gently down on top of the IEF electrode strips.

9. Place the lid on the electrofocusing chamber, connect the cables to the power supply and start IEF. Running conditions depend on the pH gradient and the length of the IPG gel strips. An appropriate time schedule for orientation is given in Table 4.

For improved sample entry, voltage is limited to 150 V, 300 V and 600 V for 1 h each, followed by 3500 V to the steady state. Current is limited to 0.05 mA/IPG strip. Optimum focusing temperature is 20°C (Görg et al. 1991).

10. After IEF, those IPG gel strips which are not used immediately for second dimension run or are kept for further reference are stored between two sheets of plastic film at -78°C up to several months (Fig. 8).

IPG-IEF for 2D electrophoresis can be simplified by the use of an integrated instrument, the IPGphor (Isalm et al. 1998). The IPGphor includes a Peltier element for temperature control (between 18°C and 25°C) and a programmable power supply (8000V, 1.5 mA). The central part of this instrument are so-called strip holders made from an aluminium oxide ceramic, in which IPG strip rehydration with sample solution and IEF are performed without further handling after the strip is placed into the strip holder (Fig. 11). The IPGphor can handle up to 12 strip holders (length 7, 11, 13 or 18 cm). The strip holder platform regulates temperature and serves as the electrical connector for the strip holders. Besides easier handling, a second advantage of the IPGphor is shorter focusing time, because IEF can be performed at rather high voltage (up to 8000 V).

The IPGphor is programmable and can store nine different programs. A delayed start is also possible, which allows the user to load the strip holders with sample dissolved in rehydration buffer in the afternoon, then automatically start IEF during the night so that IEF is finished the next morning.

2. Solubilize proteins with sample solubilization buffer containing 9 M urea, (or 7 M urea + 2 M thiourea) 2-4% CHAPS, 0.8% Pharmalytes 3-10, 15 mM DTT) and dilute the extract (protein conc.: ≈10 mg/ml) with rehydration solution (see section 4.2) (dilution: 1+1 for micropreparative runs, and 1+19 for analytical runs, respectively) to a final volume of 350 µl for 180 mm long IPG strips.

3. Put the required number of strip holders (Fig. 11) onto the cooling plate / electrode contact area of the IPGphor (Fig. 12).

4. Pipette 350 µl of sample-containing rehydration solution (for 180 mm long IPG strips) into the strip holder base. For shorter IPG strips (e.g. 110 mm) in shorter strip holders use correspondingly less liquid. Peel off the protective cover sheets from the IPG strip and slowly lower the IPG strip (gel side-down) onto the rehydration solution. Avoid trapping air-bubbles. Cover the IPG strips with 1 ml of silicone oil and apply the plastic cover. Pressure blocks on the underside of the cover assure that the IPG strip keeps in good contact with the electrodes as the gel swells.

5. Program the instrument (desired rehydration time, voltage gradient, temperature).

6. After the IPG gel strips have been rehydrated (which requires six hours at least), IEF starts according to the programmed parameters (Table 5).

7. After IEF, those IPG gel strips which are not used immediately for second dimension run or are kept for further reference are stored between two sheets of plastic film at -78°C up to several months (Fig. 8).