Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 51)

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2-D Electrophoresis Guide Methods Chapter 10: Second-Dimension SDS-PAGE
Insert the gel cassettes in the
electrophoresis apparatus and fill the
buffer chamber(s) with SDS running
buffer. SDS running buffer temperature
should be kept constant at 20°C
if the chamber design allows for
external cooling.
Connect the electrophoresis cell
to a power supply and perform
electrophoresis at 5–10 V per cm of gel
until the sample has concentrated at
the starting point of the separation gel.
Then continue with the voltage settings
recommended by the instruction
manual for the electrophoresis
system you are using.
After electrophoresis, carefully open the
cassettes and use a spatula to separate
the agarose overlay, including the IPG
strip, from the polyacrylamide gel.
Carefully peel the gel from the
cassette and place it in a container
with fixative or staining solution,
depending on the staining procedure
used (see Chapter 11).
In this stage, the equilibrated IPG strips are placed
on the top of polyacrylamide gels. This enables
smooth movement of the focused proteins into the
gel for separation by SDS-PAGE.
Reagents
Agarose solution (0.5% [w/v]): Suspend 0.5 g of
low-melting agarose (low electroendosmosis, EEO)
in 100 ml of SDS-PAGE running buffer, and dissolve
it in a boiling water bath or in a microwave oven.
Add a few crystals of bromophenol blue (or 100 µl of
1% bromophenol blue) to color the solution slightly.
The agarose solution can be aliquoted into sealed
1.5 ml or 2.0 ml plastic tubes, which can then be
melted individually in a 100°C heat block when needed.
Caution: Wear protective gloves, goggles, and a
lab coat when handling molten agarose. SDS in the
molten agarose can cause the solution to bubble
over. Molten agarose and the vessel containing it
can cause severe burns if not handled carefully.
Molecular weight standards: SDS-PAGE standards
can be applied to gels that have no reference lane.
Trim a thin filter paper to ~4 × 5 mm and pipet 10 μl of
SDS-PAGE standards onto the wick. Remove excess
solution with filter paper. Alternatively, use Precision
Plus Protein
™ standard plugs, which can be used on
vertical 2-D gels with or without a reference well.
Buffers and Solutions
This step requires the use of running buffer appropriate
for the gel chemistry you are using.
SDS-PAGE Running Buffer
(Tris-HCl and TGX
™ formulations)
Prepare sufficient 1× Tris/glycine/SDS running buffer
to run the number of gels in the system selected:
1 L of 1× Tris/glycine/SDS (25 mM Tris, 192 mM
glycine, 0.1% SDS)
Tris base
3.03 g
Glycine 14.4 g
SDS 1.0 g
Distilled or deionized H
2
O to 1 L
Alternatively, dilute 10× stock solution
(catalog #161-0732) to the desired volume.
Protocol
Perform SDS-PAGE according to the running
conditions specified for the electrophoresis system
you are using. In general:
1
1
4
3
3
4
6
2
2
5
Position the second-dimension gel
cassette so that it is leaning slightly
backwards (approximately 30°
from vertical). Use AnyGel
™ stands,
if available.
Place the equilibrated IPG strip (anodic
side on the left) onto the long plate with
the plastic backing against the plate.
Slide the strip between the plates using
a spatula to push against the plastic
backing. Ensure that the plastic backing
remains fully in contact with the long
plate and be careful not to damage the
gel with the spatula. Make sure the IPG
strip is positioned directly on top of
the second-dimension gel without any
bubbles in the interface between the
two gel surfaces.
Optional: Slip a wick soaked with
molecular weight standards or use a
Precision Plus Protein standard plug in
the slot in the gel sandwich next to or
overlapping an end of the IPG strip.
To secure the strip in place, overlay it
with molten agarose solution. Use warm
molten agarose, as hot agarose may
accelerate decomposition of the urea in
the equilibration buffer. Avoid trapping
air bubbles between the IPG strip and
second-dimension gel. Dislodge any
bubbles by tapping the plastic backing
on top of the strip.
Stand the gel upright and allow
the agarose to set for 5–10 min
before loading the gel into the
electrophoresis cell.
Protocol
Sealing IPG Strips onto SDS-PAGE Gels SDS-PAGE