Bio-Rad PROTEAN Plus Dodeca Cell - Manual (Page 11)

3.2.2 PROTEAN Plus gel cassette preparation
Cast gels in the PROTEAN Plus hinged spacer plates as directed in the PROTEAN Plus
Multi-Casting Chamber and Model 495 Gradient Former instruction manuals.
3.2.3 PROTEAN II Ready Gel precast gel and PROTEAN II glass plate
Preparation
The PROTEAN II Ready Gels with
the IPG comb and the 20 x 20 cm
PROTEAN II glass plates are compatible
with the PROTEAN Plus Dodeca cell. To
prevent current leaks, the side of the gel
placed at the bottom of the tank MUST be
greased with high vacuum grease. Prior to
loading an IPG strip, dry the edge of the
cassette and then heavily coat the entire
edge of the glass plates and spacer with
high vacuum grease using a spatula
(Figure 3.2.3A).
For further information on precast
gels refer to the PROTEAN II Ready Gel
user manual for complete precast gel
instructions.
Fig. 3.2.3A. Applying grease to PROTEAN II handcast
or Ready Gel precast gel cassettes.
3.3 IPG Strips Preparation and Loading
See the Ready Strip ™ IPG strips instruction manual for proper first-dimension
preparation.
Position the slab gel cassette so that it is leaning slightly backwards at a small angle, the
6-row AnyGel Stand was designed for this application (Catalog # 165-5131). The best way
to apply a standard lane is using a piece of filter paper, the precut electrode wicks
(Catalog # 165-4071) that are used with the PROTEAN IEF cell work well. Pipette 10 µl of
Precision unstained protein standards (Catalog # 161-0363) onto a single wick and allow the
liquid to dry. Using forceps place the wick on the slab gel, either in a reference well or just
sitting on top of the slab gel, make sure to leave enough room for the strip. Prepare a 50–100 ml
graduated cylinder filled with running buffer, then while holding the IPG strip vertically with
forceps, dip the IPG strip into the buffer. Place the IPG Strip onto the long plate with the
plastic backing against the plate. Slide the strip down in between the plates using a thin
spatula and pushing against the plastic backing. Be careful not to damage the gel with the
spatula. Make sure the IPG strip is positioned directly on top of the slab gel. To secure the strip
in place overlay with 1.0% agarose prepared in SDS-PAGE running buffer (a small amount
of Bromophenol Blue can be added to the agarose overlay in order to track the ion front).
Bubbles may form under or behind the strip when adding the agarose overlay, these may
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