Biology Reference
In-Depth Information
Table 3
Parameters recommended by GE Healthcare for isoelectric
focusing of preparative saturation DIGE samples
Step
Power (V)
Ramp
Duration (h)
1
150
Step and hold
4.0
2
300
Step and hold
3.0
3
1,000
Gradient
6.0
4
8,000
Gradient
1.0
5
8,000
Step and hold
6.0
6
500
Step and hold
Up to 48.0
Current should be below 75
μ
A/strip, and temperature should be adjusted
to 25°C
Generate a fl uorescence Cy3 image from the preparative gel by fol-
lowing the instructions of the scanner's manufacturer. Use the
same instrument as used for the analytical gels. Make sure no dis-
tortions of the gel can occur between the start of scanning and the
end of spot picking, e.g., by using an appropriate gel frame.
Carefully match the spots in the preparative gel with the spots
from an analytical gel, preferably using the readout representing
the IPS. For spots to be identifi ed by mass spectrometry, a very
careful manual inspection for correct matching is indispensable.
Print out a true to scale image of the Cy3 readout and mark all
spots to be picked for identifi cation.
3.8.1. Visualization
and Assignment of Spots
Spots can either be picked by a spot-picking robot or manually. For
spot picking by a robot, the following procedure is recommended:
3.8.2. Spot Picking
1. Export the
x-
and
y
-coordinates of relevant spots from DeCyder
and import these into the software of the spot-picking robot.
Refer to the robot's manual for this procedure.
2. After picking, perform a rescan of the Cy3 image to control if
spots have been hit correctly by the spot excision device.
3. In addition, a subsequent Coomassie staining may be helpful
(see Notes 19 and 20).
For manual spot picking, the following procedure is recommended:
1. Stain the gel with colloidal Coomassie (freshly prepared and
keratin-free) to generate visible spots (see Notes 19 and 20).
2. Carefully align the gel to the true to scale printout, using char-
acteristic spots visible in both the Cy3 and the Coomassie
stained gel image.
3. Pick spots using your preferred spot-picking device.
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