Biology Reference
In-Depth Information
Table 2
IPGphor programme used in the isoelectric focusing
of mouse liver lysates
Stage
Step or gradient
Voltage (V)
Duration or kV-hours
S1
Step
150
2 h
S2
Step
500
2 h
S3
Gradient
1,000
3 h
S4
Gradient
8,000
5 h
S5
Step
8,000
96,000 kVh
S6
Step
500
2,000 kVh
can be stopped at any stage during S6, and the strips removed
promptly before they are processed further (see Note 8):
1.
Level the IPGphor II and ensure that the ceramic tray is com-
pletely clean and dry (see Note 9).
2.
Place the ceramic tray onto the IPGphor II and carefully add
approximately 108 mL of DryStrip cover fl uid, ensuring that
the fl uid is evenly distributed across the lanes.
3.
Using two pairs of tweezers, carefully remove the IPG strips
from the rehydration tray and drain off any surplus cover fl uid
onto a lint-free wipe. Ensure that the strip is placed gel-side
up—the serial nos. should not be readable.
4.
Place each strip, gel-side up, into a lane of the ceramic tray
so that the end of the strip marked with “+” is furthest away
from you.
5.
Dampen each wick with 150 mL of deionised water and place
them at both ends of each IPG strip, so that they slightly
overlap the ends of the gel (see Note 10).
6.
Place each electrode assembly on top of the wicks at either end
of the IPG strips. The assembly should make electrical contact
with the IPG strips through the wicks, and with the IPGphor.
7.
Add more DryStrip cover fl uid to the tray so that the strips
are completely covered, close the lid and protect the strips
from light.
8.
Set the IPGphor to run at 20°C and 75 mA/strip. The pro-
gramme detailed in Table
2
has been successfully used for
mouse livers.
9.
Start the focusing programme (see Table
2
) and enter the
number of IPG strips when prompted.
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