Bio-Rad Model 111 Mini IEF Cell - Manual (Page 21)

3. Lightly moisten the Graphite Electrodes with water. Turn the gel with the adsorbed samples
upside down and place it directly on top of the electrodes. Position the gel carefully the first
time as re-positioning may damage the gel surface. Do not remove the glass plate from the
gel/gel support backing because its weight insures good contact between the gel and the
electrodes.
4. Carefully slide the lid back onto the Model 111 Mini IEF Cell. Plug the power cab cell into a
power supply able to generate 500 V constant voltage. The cell is now ready to separate
samples.
6.7 Run Conditions
1. Focusing is carried out under constant voltage conditions in a stepped fashion. Begin focusing
at 100 V for 15 minutes.
2. Increase voltage to 200 V for 15 minutes.
3. Finally, increase the voltage to 450 V for an additional 60 minutes.
Note: Step increases of voltage are necessary to prevent overheating and
subsequent dehydration of the gel. Failure to follow this procedure will result in poor
resolution.
4. A good way to monitor the progress of an isoelectric focusing experiment is to observe the
migration of visible marker proteins of known pI to their isoelectric points. The IEF Standards
(catalog number 161-0310) provide eight natural proteins, including four visible proteins that
are clearly discernable during an IEF run. These markers are useful with all non-denaturing
IEF buffer systems.
5. As the focusing nears completion, the current will decrease substantially. This is a general
indication that the focusing is near completion.
6.8 Sample Detection
1. After the focusing is complete, turn off the power supply and disconnect the power cables
from it.
2. Slide the lid off the Model 111 Mini IEF Cell and carefully remove the gel from the Graphite
Electrodes. Rinse the electrodes with water and wipe them gently with a tissue. Replace the
electrodes in the cell.
3. Place the gel/gel support film (Glass Plate is removed at this point) into fixative solution for
15 minutes.
Fixative Solution
30% methanol
5% trichloroacetic acid (TCA)
3.5% sulfosalicylic acid (SSA)
4. To insure a clear gel background, take the agarose gel directly from the fixative solution to a
95% ethanol bath. Immerse the gel in ethanol for 30 minutes with occasional swirling.
5. After the ethanol wash, place the gels on a level surface. Soak one piece of filter paper in
ethanol and place it on top of the gel. Then place additional dry filter paper (8-10 sheets) or
folded paper towels on top of the ethanol-soaked paper. Press the gel with a 1 kg weight for
30 minutes.
6. After pressing, dry the gel completely with an air blow dryer or under a laboratory hood’s fan.
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