Bio-Rad Criterion™ TBE-Urea Precast Gels - Manual (Page 35)

2-D Electrophoresis
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11.1 Introduction
Criterion ™ precast gels are available for second-dimension PAGE in 2-D electrophoresis workflows.
The IPG-well gels accommodate 11 cm IPG strips. Criterion ™ Any kD ™ gels are particularly well suited
for 2-D electrophoresis applications.
The transition from first- to second-dimension gel electrophoresis involves:
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Equilibration of the resolved IPG strips in a reducing buffer containing SDS
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Placing the IPG strip on top of the second-dimension gel
11.2 Equilibration
Equilibration ensures that proteins in the IPG strips are coated with SDS and that cysteines are reduced
and alkylated. Use the equilibration protocols (bulletin 4110009) and buffers in the ReadyPrep
™ 2-D
starter kit (catalog #163-2105), or other protocols and buffers used with Tris-HCl gels.
11.3 Agarose Overlay
Place the equilibrated IPG strip into the IPG well of the Criterion gel and overlay it with molten agarose to
ensure good contact between the strip and gel.
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Criterion TGX ™ and Tris-HCl gels: prepare 0.5% low-melt agarose (catalog #161-3111),
0.003% bromophenol blue (catalog #161-0404) in 1x Tris/glycine/SDS running buffer.
(Alternatively, use ReadyPrep overlay agarose, catalog #163-2111)
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Criterion ™ XT gels: prepare 0.5% low-melt agarose (catalog #161-3111), 0.001%
bromophenol blue (catalog #161-0404) in appropriate 1x XT running buffer
1. Following equilibration, place the IPG strip, gel side up, on the back plate of the Criterion gel, above
the IPG well. The “+” and pH range on the IPG strip should be on the left.
2. Using forceps, push the strip into the IPG well, taking care to not trap air bubbles under the strip.
Push on the backing of the strip, not on the gel.
3. Using a disposable pipet, add overlay agarose to the IPG well. Fill the well to the top of the inner
plate. Dispense rapidly, as overlay agarose solidifies quickly. To avoid bubbles, tilt the cassette
slightly to allow bubbles to escape. Push gently on the plastic backing of the strip to free any
trapped bubbles.
11.4 Second-Dimension Electrophoresis
Place the cassettes into the Criterion cell and start the run using the run conditions for SDS-PAGE.
Use the migration of the bromophenol blue in the overlay agarose to monitor the progress of the run.
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