Symbiotic Fungi: Principles and Practice (Soil Biology)

15 Functional Genomic of Arbuscular Mycorrhizal Symbiosis 255
Note 10: Overlay the gels with mineral oil to control the correct positioning of the
sample cups
4. Carry out the IEF at 20 C with the Multiphor II system (AB) as recommended by
the manufacturer
5. Store the strips in clean plastic trays at 80 C, or equilibrate them according to
Görg et al. (1987) for immediate separation on SDS-PAGE.
In order to determine the Mw and pI of the polypeptides separated in the gels, add
10 ml of standard proteins (2-D, Bio-Rad, AB, etc.) for co-migration with samples.
Alternatively, the standard proteins may be loaded on a small piece of Whatman
paper placed on the border of the 2D gels.
15.4.5.4 Electric Focusing
1. For analytical separations, 100 mg of total or cytosolic proteins are usually
loaded and focused at 20 C for 50 kVh using a gradually increasing voltage.
2. For micropreparative analysis, up to 600 mg of proteins can be loaded and the
focusing extended to 71 kVh (Valot et al. 2005).
3. Carry out the focusing at 20 C with the Multiphor II system (AB) under mineral
oil as follows:
l Pre-electrophoresis with successive steps (150, 300 and 1,500 V)
l Then electrophoresis at 3,500 V overnight at 20 C according to the following
programme
1/1 150 V 1 mA 5 W 0:01 h
1/2 150 V 1 mA 5 W 0:30 h
1/3 300 V 1 mA 5 W 0:01 h
1/4 300 V 1 mA 5 W 1:00 h
1/5 1,500 V 1 mA 5 W 1:00 h
1/6 1,500 V 1 mA 5 W 1:00 h
1/7 3,500 V 1 mA 5 W 3:00 h
1/8 3,500 V 5 mA 5 W 20:00 h
l The last steps needs at least 12 h for analytical gels and 18 h for the micro
preparative ones
15.4.5.5 IEF with the IEF Cell
1. Connect the IEF Cell. Launch the ‘‘rehydratation’’ program: that will allow the
system to equilibrate at 20 C.
2. Place the electrode strips (previously wetted with milli Q water) over the platinum
electrodes at the extreme part of each channel of the tray (12 maximum).