Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 13)

4.4 Completion of IEF.
1. When the electrophoresis run has been completed, remove the IPG strips from the focus-
ing tray and transfer them gel side up into a new or clean, dry disposable rehydra-
tion/equilibration tray which matches the length of the IPG. Hold the strips vertically with
the forceps and let the mineral oil drain from the strip for ~5 seconds before transfer.
Maintain the IPG strips in the same order as in the focusing tray.
2. Proceed to section 4.5 to stain two of the strips, and then proceed to section 4.6 with the
remaining strips. Alternatively, the tray containing the remaining IPG strips can be covered,
wrapped in plastic wrap and placed at -70ºC for storage. The second-dimension SDS-
PAGE gels can then be run at a later time.
4.5 Staining IPG Strips with IEF Stain or BioSafe Coomassie Stain.
1. Transfer 2 of the IPG strips to a clean, dry piece of blotting filter paper with the gel side
facing up.
2. Thoroughly wet a second filter paper of the same size with nanopure water. Carefully lay
the wet filter paper onto the IPG strips. Press firmly over the entire length of the strips. Do
NOT squish the gels. When finished, carefully “peel” back the top filter paper. This blotting
step removes mineral oil on the surface of the IPG, thereby reducing background staining
and generally improving the staining of the IPG strips.
3. Transfer the 2 IPG strips to a staining tray containing approximately 50 ml of Bio-Safe
Coomassie stain or Bio-Rad’s IEF stain.
4. Place the tray onto a rocking platform or orbital shaker for 1 hour.
5. Destain the IPG strips twice for 10 minutes each. For Bio-Safe stain use 20 mM Tris-HCl,
pH 8.8. For IEF stain use destain solution (catalog #161-0438). Complete destaining of
the IPG strips may take several hours. Changing the destain solution several times can
accelerate this process.
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After IEF it is sometimes helpful to remove a subset of the IPG strips and stain them with either
IEF stain or Bio-Safe Coomassie stain to assess the quality of the focusing step. A procedure
for staining IPG strips after the IEF step is described in section 4.5. Staining two of the six strips
focused is recommended. However, this IEF stain step is optional.
For 7 cm or 11 cm strips, after completion of the focusing step it is recommended to remove
two of the focused strips and stain them following section 4.5, steps 1-4. During the staining
step (section 4.5, step 4), a 1 hr incubation in staining solution, it is recommended to equilibrate
and mount the remaining strips on the second-dimension gels (section 4.6). Once the SDS-
PAGE gels are running, return to section 4.5, step 5 to destain the first-dimension stain.
For 17 cm strips, after the completion of the focusing step it is recommended to remove two of
the focused strips and stain them following section 4.5. The remaining strips (gel side up in the
disposable rehydration/equilibration tray per section 4.4, step 1) can be covered with the tray
cover, and then wrapped in plastic wrap and placed at -70ºC for storage. The second-dimen-
sion SDS-PAGE gels can then be set up and run on Day 2.