Bio-Rad Mini-PROTEAN® TGX™ Precast Gels for 2-D Electrophoresis - Manual (Page 28)

2-D Electrophoresis
9
9.1 Introduction
Mini-PROTEAN ® precast gels are available for second-dimension PAGE in 2-D electrophoresis
workflows. The IPG-well gels accommodate 7 cm IPG strips. Mini-PROTEAN ® TGX Any kD ™ gels are
particularly well suited to 2-D electrophoresis applications.
The transition from first-to second-dimension gel electrophoresis involves:
n
Equilibration of the resolved IPG strips in an SDS-containing, reducing buffer
n
Placing the IPG strip on top of the second-dimension gel (agarose overlay)
9.2 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 411009) and buffers in the ReadyPrep
™ 2-D
starter kit (catalog #163-2105), or other protocols and buffers used for Tris-HCl gels.
9.3 Agarose Overlay
Place the equilibrated IPG strip into the IPG well of the gel and overlay it with molten agarose to ensure
good contact between the strip and gel.
1. Prepare 0.5% low-melt agarose (catalog #161-3111), 0.003% bromophenol blue (catalog #161-0404)
in 1x Tris/glycine/SDS running buffer (or use ReadyPrep overlay agarose, catalog #163-2111).
2. Following equilibration, place the IPG strip, gel side up, on the back plate of the gel, above the IPG
well. The “+” and pH range on the IPG strip should be on the left.
3. 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.
4. Using a disposable pipet, apply overlay agarose into 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 backings of the strip to free any
trapped bubbles.
9.4 Second-Dimension Electrophoresis
Place the cassettes in to the Mini-PROTEAN ® Tetra 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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