Bio-Rad PROTEAN® i12™ IEF System - Manual (Page 13)

Chapter 2 Basic Operation
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2.2.1 IPG Strip Rehydration in the Rehydration/Equilibration Tray Followed by IEF
Rehydration IEF
2.2 IPG Strip Rehydration and Sample Application
The choice of rehydration and sample loading method (in-gel sample loading or cup loading) and IPG strip
configuration (gel-side down or gel-side up) dictates the workflow for the run. The first step is to rehydrate
the IPG strips in rehydration solution, with or without sample (Table 2.1).
IPG strips can be rehydrated in either the i12 rehydration/equilibration trays or the i12 focusing tray:
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For rehydration in rehydration/equilibration trays, see Section 2.2.1
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For rehydration in the focusing tray, see Section 2.2.2. Rehydration in the focusing tray with in-gel sample
application can be programmed as part of the IEF run
See Appendix B, Sample Loading Methods and Running Configurations, for guidelines for
selecting the sample loading method and IPG strip configuration.
Table 2.1. Rehydration volumes, sample loads, and mineral oil volumes. The values listed are recommendations. Optimum
sample load depends on sample type. See Appendix A, Reagent and Sample Preparation, for more details.
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Rehydration Solution 125 μl 200 μl 300 μl 315 μl 450 μl
Protein Load
Coomassie (Brilliant) Blue
50–100 µg 100–200 µg 200–400 µg 200–400 µg 400–800 µg
Fluorescent stains 5–100 µg 20–200 µg 50–400 µg 50–400 µg 80–800 µg
Silver stains 5–20 µg 20–50 µg 50–80 µg 50–80 µg 80–150 µg
Mineral Oil 4 ml 5 ml 7 ml 7 ml 9 ml
For the rehydration step:
1. Pipet the rehydration solution (with or without sample, see Table 2.1 for volumes and protein loads) along
the center of the channel(s) of the i12 rehydration/equilibration tray. Take care to not introduce air bubbles
when expelling the solution.
2. Using forceps, remove the cover sheet from the IPG strip, then gently place the IPG strip gel-side down
onto the solution in the channel. Move the IPG strip back and forth slightly to ensure that the solution is
distributed along the length of the IPG strips. Take care to not trap air bubbles beneath the IPG strip.