Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 46)

Fig. 9.2. Rehydration in the focusing tray.
Rehydration IEF
With sample
(passive or active)
With gel-side down
IPG strips remain
in focusing tray
88 89
2-D Electrophoresis Guide Methods Chapter 9: First-Dimension IEF with IPG Strips
Position the electrode assemblies in
the channels of the focusing tray and
press down on the green tabs to snap
the electrode assemblies into place.
Recommended: Wet the rectangular
(gel-side down) wicks with distilled or
deionized water and blot off excess
water. Use two wicks per IPG strip:
place a wick on top of each electrode.
Using forceps, place the rehydrated
IPG strips gel-side down in the
channels of the focusing tray.
Position the positive (+) ends of the
IPG strips against the positioning
stops in each channel.
Place the focusing tray on the Peltier
platform and connect the electrodes
to the instrument.
Overlay each IPG strip with
mineral oil (see Table 9.1 for
recommended volumes).
Place the IPG strip retainers on top of
the IPG strips at both the positive and
the negative ends. Without IPG strip
retainers in place, gases formed
during electrolysis may lift IPG
strips off the electrodes, interrupting
electrical contact.
Select or program the protocol(s)
and start the run.
Position the electrode assemblies
in the focusing tray.
Pipet the rehydration solution containing
the protein sample along the center of
the channel(s) of the focusing tray
(see Table 9.1 for recommended volumes
and protein loads). Do not introduce air
bubbles when expelling the solution.
Using forceps, remove the cover sheet
from the IPG strip, then gently place
the IPG strip gel-side down onto the
sample in the channel of the tray.
To ensure even rehydration, move the
IPG strip back and forth slightly to
distribute the solution along its length.
Check that no bubbles are trapped
beneath the strips and that some
rehydration solution extends beyond
the electrode contacts.
Place the focusing tray with the IPG
strips on the Peltier platform and
connect the electrodes to
the instrument.
Immediately overlay each IPG strip with
mineral oil to prevent evaporation and
precipitation of urea during rehydration.
Apply the mineral oil to both ends
of the channel and allow it to flow
toward the middle. See Table 9.1 for
recommended volumes of mineral oil.
Using forceps, place the rehydrated IPG
strips gel-side up in the channels of
the focusing tray. Position the positive (+)
end of the IPG strips against the
positioning stops in each channel.
Recommended: Wet the gel-side
up electrode wicks (notched) with
deionized water and blot off excess
water. Use two wicks per IPG strip:
place a wick at each end of each
IPG strip.
Position the electrode assemblies in
the focusing tray and press down on
the green tabs to snap the electrode
assemblies into place. Place the
focusing tray with the rehydrated IPG
strips on the Peltier platform, and
connect the electrodes to
the instrument.
Prepare the sample cup assembly by
placing the sample cups into the slots
of the sample cup holder corresponding
to the channel with the rehydrated
IPG strip.
Clamp the sample cup assembly onto
the edges of the focusing tray, on top
of the IPG strips and next to either
electrode. Placement depends on the
pH gradient and the sample. In general,
focusing is most effective towards
the end of the IPG strip opposite the
site of the sample cup placement.
Use anodic sample cup placement
when using basic pH ranges or when
resolution of basic proteins is desired.
Load 25–250 μl of sample into the
sample cups (larger volumes of dilute
samples of up to 400 μl may be loaded).
Overlay both the sample in the sample
cup and the IPG strip with mineral oil.
Select or program the protocol(s)
and start the run.
Position the IPG strip retainers on top
of the IPG strips at both the anode
and the cathode to maintain electrical
contact with the IPG strips. Without the
IPG strip retainers, gases formed during
electrolysis may lift the IPG strips
off the electrodes, interrupting
electrical contact.
Rehydration in the focusing tray with in-gel
sample application can be programmed
as a part of the IEF
run or be performed separately.
To program rehydration as part
of the run:
a.
Select or program the protocol(s)
for the lanes containing IPG strips
b. Program the global rehydration
conditions. If electrode wicks are
used, include a post-rehydration
pause to insert electrode wicks
when the rehydration step is
completed
c. Start the run
For rehydration not programmed as part
of the run, leave the tray on the Peltier
platform or on a level bench overnight
(11–16 hr) for complete rehydration.
Start the run (perform IEF).
IEF with Gel-Side Down
The following protocol is for IPG strips that have
been rehydrated in the presence of sample
(in-gel sample loading).
Protocol
Cup Loading (IEF with Gel-Side Up)
This protocol is for IPG strips that have been
rehydrated in the absence of sample. Sample cups
offer an alternative method of sample loading, and
their use can often improve resolution, especially at
extreme pH ranges. The PROTEAN i12 sample cup
assembly consists of a sample cup holder that holds
1–12 disposable sample cups.
Protocol
IPG Strip Rehydration in the Focusing Tray Followed by IEF
For rehydration and IEF in the focusing tray, place the IPG strip gel-side down on top of the rehydration solution
in the focusing tray. Rehydration can be programmed as a part of the IEF run, and the protocols can be
programmed next. Alternatively, the strips can be rehydrated independently and the protocol(s) started
when most convenient.
Protocol
1
1
1
6
3 3
3
4
4
4
5
5
5
8
6
6
7
7
2
2
2
7
Performing IEF
(contd.)