Bio-Rad PROTEAN II XL Cell - Manual (Page 33)

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Embedding of First Dimension onto Second Dimension
Note: When handling gels, it is advisable to wear gloves. First dimension gels tend to be sticky to the touch
and will tear easily.
1. Use of a beveled short glass plate (catalog number 165-1827 for 16 cm cell and
165-1828 for 20 cm cell) in the second dimension slab sandwich will greatly improve the ease of
embedding the tube gel on top of the slab gel. Rinse the top of the completed second dimension SDS
slab gel thoroughly with distilled water, and drain off excess water. Attach clamp assemblies to cooling
core.
2. Using a spatula, direct the gel from the Parafilm to the bevel at the top of the inner glass plate, starting
at one side and proceeding across the gel. Place a few drops of SDS electrode buffer along the top of
the tube gel.
3. Use a spatula to seat the tube gel on the slab gel. Check that the tube gel is in contact with the slab gel
over its entire length. Be sure to remove all air bubbles that are between the tube gel and the slab gel.
By placing the tube gel directly on top of the second dimension slab gel, between the glass plates, no
agarose overlay is necessary. If the tube gel diameter is greater than the second dimension slab gel
thickness, an agarose overlay may be necessary to insure good contact and to prevent the tube gel
from slipping off the slab gel. The agarose overlay is 1% agarose in 1x stacking gel buffer, diluted 1:4
(see Section 14.1, solution C).
Note: If you would like to apply molecular weight standards to the second dimension, we recommend
using a 2-D comb for casting the stacking gel. As an alternative, if a stacking gel is not desired, you can
make a tube gel with a mixture of agarose and Bio-Rad standards. Then simply cut the agarose into
pieces, and load a piece directly on top of the second dimension slab gel in tandem with the IEF tube gel.
4. Electrophorese the SDS slab gel as in Section 7.
5. Remove the gels as in Section 9.
6. Stain the gels as in Section 14.5.
Note: Tube gels may be frozen for future use or applied directly to a second dimension slab gel. To
freeze a tube gel, place the gel lengthwise in a stoppered tube in an EtOH:dry ice bath. Once frozen,
gels can be stored at -20 °C.