Biology Reference
In-Depth Information
7.
Combine the three differentially labeled samples into a single
microfuge tube and mix. One of these samples should be the
pooled internal standard.
8.
Add a volume of rehydration buffer (7 M urea, 2 M thiourea,
4% (w/v) CHAPS, with 1% ampholytes, 20 mM DTT) and
leave on ice for at least 10 min. The samples are now ready for
the fi rst dimension isoelectric focusing step. Once the rehydra-
tion buffer has been added, it is recommended that the sample
is run immediately after at least 10 min on ice.
9.
Load up to 100 mL of sample into the bottom of the loading cup.
10. Put the clear plastic strip cover onto the strip holder. The strip
holder is now ready to be loaded onto the IPGphor IEF unit.
11. For cup loading, the following parameters were used: 20°C
50-75 mA/strip, applying an increasing voltage as follows:
300 V for 3 h, gradient increase from 300 to 1,000 V for 6 h,
gradient increase from 1,000 to 8,000 V for 3 h, and a fi nal
step increase to 8,000 V for 3 h.
12. When focusing is complete, remove the strip, drain off the
mineral oil on a paper towel, and place it onto a strip holder
with gel side down and cover the acidic end of the strip with
parafi lm. You can store the strip at −20°C in the dark or con-
tinue with the second-dimension SDS-PAGE.
3.8. Second-
Dimension SDS-PAGE
1.
Add 4 mL of reduction solution to the strip, cover the basic
end of the strip with parafi lm, and incubate with agitation for
10 min.
3.8.1. Equilibration
2.
Open the basic end of the strip and discard the solution, drain-
ing it off on a paper towel. Then, add 4 mL of alkylation solu-
tion, cover, and incubate in the same way as described above.
After draining off the alkylation solution, briefl y rinse the
Immobiline DryStrip by submerging it in SDS electrophoresis
running buffer.
1.
The second dimension is run on a 12.5% acrylamide gel with
0.2% (w/v) SDS (see Subheading
2.7
, item 9
,
and Note 19).
When pouring the gel, remember to take into account that a
1-cm gap should be left at the top of each gel to allow the IPG
strip to be loaded.
3.8.2. SDS-PAGE
2.
Following IPG strip equilibration, the gel should be overlaid
with hot agarose solution (see Subheading
2.7
, item 1), and
the strip should be pushed through the still liquid agarose.
3.
The amount of SDS electrophoresis buffer required depends
on the gel tank used. For the lower buffer tank of the Ettan
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