Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 50)

Table 10.1. Recommended equilibration volumes.
IPG Strip Length
7 cm 11 cm 17 cm 18 cm 24 cm
Equilibration buffer 1 2.5 ml 4 ml 6 ml 6 ml 8 ml
Equilibration buffer 2 2.5 ml 4 ml 6 ml 6 ml 8 ml
10 min is recommended for each equilibration step.
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2-D Electrophoresis Guide Methods Chapter 10: Second-Dimension SDS-PAGE
■ ■
Use 5–10 V per cm of gel for 10 to 30 min during
sample entry (until the sample has concentrated
at the starting point of the separation gel).
Then continue with the voltage setting
recommended in the instruction manual for
the electrophoresis system you are using
■ ■
Use the voltage setting recommended in the
instruction manual for the electrophoresis system
you are using; excessive voltage leads
to decreased resolution and distortions
■ ■
When running multiple cells, use the same
voltage for multiple cells as you would for one cell.
Be aware that the current drawn from the power
supply will double with two — compared to one —
cells. Use a power supply that can accommodate
this additive current and set the current limit high
enough to permit this additive function
■ ■
To maximize reproducibility, maintain the
temperature of the electrophoresis buffer at
about 20°C with the help of a recirculating cooler
Tips for SDS-PAGE
■ ■
Ensure that gels have the same composition by
either using precast gels, which are manufactured
in lots and so are virtually identical, or hand
casting the gels at the same time in a multi-
casting chamber
■ ■
Save time by preparing the overlay solution
and running buffers during the 10 min
equilibration incubations
■ ■
Vertical streaking on second-dimension gels is
often caused by gaps between the IPG strips and
the gels. Ensure that the second-dimension gel
has a straight and level top edge, and that the
IPG strip is in direct contact with the gel along its
entire length
■ ■
When preparing running buffers, make the solution
as specified in the protocol and do not titrate to a
pH. The ion balance is set by the concentration of
reagents; adjusting the pH alters this balance and
leads to undesirable results
■ ■
Do not reuse running buffers
Place one IPG strip gel-side up in each
channel of a rehydration/equilibration
tray, and fill the channels with
the recommended volume of
equilibration buffer.
Incubate with gentle agitation for
10 min, then decant.
Fill the channels with the recommended
volume of equilibration buffer 2,
and incubate again for 10 min.
After equilibration, remove the IPG
strips and briefly rinse with the
SDS-PAGE running buffer you will be
using. This step rids the IPG strip of
excess iodoacetamide and serves to
lubricate the IPG strip for placement
on the second dimension.
Equilibrate the IPG strips twice, each time for 10 min,
in two different equilibration buffers. Use disposable
rehydration/equilibration trays for this purpose.
Reagents
Tris-HCl buffer (25 ml)
1.5 M Tris-HCl (pH 8.8)
Dissolve 4.55 g of Tris base in ~20 ml of deionized
or distilled H
2
O. Adjust the pH of the solution with
diluted HCl and adjust the volume to 25 ml with
distilled or deionized H
2
O.
Equilibration stock buffer (500 ml)
6 M urea, 30% (w/v) glycerol, 2% (w/v) SDS in 0.05 M
Tris-HCl buffer, (pH 8.8). Pre-prepared equilibration
buffers can also be purchased.
Combine 180 g of urea, 150 g of glycerol, 10 g of SDS,
and 16.7 ml of Tris-HCl buffer. Dissolve in deionized
distilled H
2
O and adjust the volume to 500 ml.
Store frozen.
Equilibration buffer 1 (10 ml)
Add 100 mg of DTT to 10 ml of equilibration
stock buffer.
Equilibration buffer 2 (10 ml)
Add 400 mg of iodoacetamide to 10 ml of
equilibration stock buffer.
Protocol
1
2
3
4
IPG Strip Equilibration