Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 17)

Chapter 2 Basic Operation
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2.2.2 IPG Strip Rehydration in the Focusing Tray Followed by IEF
Rehydration can be programmed as a part of the run, followed by the IEF protocols. Alternatively, the IPG
strips can be rehydrated independently and the protocol(s) started when most convenient.
Rehydration IEF
1. Position the electrode assemblies in the focusing tray as described in Section 2.1.1.
2. Pipet the rehydration solution containing the protein sample along the center of the channel(s) of the
focusing tray (see Table 2.1 for recommended volumes and protein loads). Do not introduce air bubbles
when expelling the solution.
3. Using forceps, remove the cover sheet from the IPG strip, then gently place the IPG strip gel-side down
onto the sample in the channel of the tray. To ensure even rehydration, move the IPG strips back and
forth slightly to distribute the solution along the lengths of the IPG strips. Check that no bubbles are
trapped beneath the IPG strip and that some rehydration solution extends beyond the electrode contacts.
4. Place the focusing tray with the IPG strips on the Peltier platform and connect the electrodes to the
instrument (see Section 2.1.2).
5. Immediately overlay each IPG strip with mineral oil to prevent evaporation and precipitation of urea during
rehydration. Apply the mineral oil to both ends of the channel and allow it to flow toward the middle of the
channel. See Table 2.1 for recommended volumes of mineral oil.
6. Position the IPG strip retainers on top of the IPG strips at both the anode and the cathode to maintain
electrical contact with the IPG strips during IEF. Without the IPG strip retainers, electrolysis gasses may lift
IPG strips off of the electrodes, interrupting electrical contact.
For rehydration not programmed as part of the run, leave the tray on the Peltier platform or on a
level bench overnight (12–18 hr) for complete rehydration.
Global rehydration conditions are applied to all IPG strips in a run.
Place IPG strip retainers after the focusing tray has been placed into the instrument to avoid
movement of the strip retainers.
7. Rehydration in the focusing tray with in-gel sample application can be programmed as a part of the IEF
run or be performed separately. To program rehydration as part of the run:
a. Select or program the protocol(s) for the lanes containing IPG strips (see Section 3.1).
b. Program the global rehydration conditions in the Run Settings screen (see Section 3.2). If electrode
wicks are used, include a pause to insert electrode wicks when the rehydration step is completed.
c. Start the run (see Section 2.3, Starting the Run).