Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 10)

4 Overlay each of the strips with 2 to 3
ml of mineral oil to prevent evaporation
during the rehydration process. Add
the mineral oil slowly, by carefully drip-
ping the oil onto the plastic backing of
the strips while moving the pipet along
the length of the strip.
5 Cover the rehydration/equilibration tray
with the plastic lid provided and leave
the tray sitting on a level bench
overnight (11-16 hr) to rehydrate the
IPG strips and load the protein sample.
This is the final step of day 1.
4.3 Isoelectric Focusing.
1 Place a clean, dry PROTEAN IEF focusing tray the same size as the rehydrating IPG strips
onto the lab bench.
2 Using forceps, place a paper wick at both ends of the channels covering the wire electrodes.
Use channels with the same numbers as those used during rehydration. As shown in Figure
5, channel numbers 1, 2, 3, 10, 11, and 12 contain electrode wicks.
3 Pipet 8 µl of nanopure water (provided) onto each wick to wet them as shown in Figure 6.
Readjust their position if necessary.
4 Remove the cover from the rehydration/equilibration tray containing the IPG strips. Using
forceps, carefully hold the strip vertically for about 7 to 8 seconds to allow the mineral oil
to drain (Figure 7), then transfer the IPG strip to the corresponding channel in the focusing
tray (maintain the gel side down) (Figure 8). Repeat for all the strips.
Electrode wicks are important as recepta-
cles for salts and other non-amphoteric
constituents of the sample, and they
improve the quality of the results.
8
Mineral oil prevents evaporation of the sam-
ple during rehydration, thus preventing pre-
cipitation of the urea. IPG strips can be left
to rehydrate for up to 1 hr before adding the
oil. This simplifies overlaying the strips with
oil as most of the sample is now absorbed
into the strip.
Fig. 5. Placement of paper wicks over
electrode at each end of channel.