Biology Reference
In-Depth Information
6. Optionally, the labelling reaction can be checked on a small
SDS-PAGE on some of the samples before running the com-
plete series of DIGE.
7. When the mineral oil covers the gels, check that there is no
leakage (no oil in the sample cups).
8. SDS denatures proteins and forms negatively charged protein-
SDS complexes.
9. IAA alkylates thiol groups on proteins, preventing their reoxi-
dation during electrophoresis. Protein reoxidation during elec-
trophoresis can result in streaking and other artifacts. IAA also
alkylates residual DTT to prevent point streaking and other
silver-staining artifacts.
10. In the above text, if it is not mentioned otherwise, “water”
means double-distilled water.
11. If it is not possible to scan all the gels at the same day, they can
be stored at 4 °C for 48 h without variation in the signal and
the position on the gel. For longer storage periods, the gels
have to be fi xed in fi xing solution and then kept at 4 °C.
Before scanning, leave the gels on the bench for a while to
reach room temperature (as the temperature impacts the sig-
nal intensity).
12. Preparative gels, containing high amount of proteins, can be
prepared separately for the identifi cation of proteins.
We recommend either to prepare an important amount of
proteins coming from the different conditions (e.g., with the
same amount as for internal standard) or to run one prepara-
tive gel per sampling condition.
In this case, the labelling step is omitted and the gel is
stained with Coomassie or another fl uorescent solution (Sypro
Ruby, Lava Purple, or others). At least 300
g have to be
loaded on the IPG strips, and the time for IEF has to be
extended (for a 24-cm gel, pH 4-7, the recommended value
to reach is 90,000 V h). It is also recommended to use Bind-
Silane-coated glass plates to facilitate the picking (by avoiding
shrinking or deformation of the gels).
13. Care is needed with the microplates; digestion carried out in
microplates from several suppliers has resulted in the presence
of polymers in the mass spectra, rendering the MS/MS spectra
unreadable.
14. If the preparative gels are stained with fl uorescent dyes and/or
used for picking with the Ettan Spot picker or the Spot
Handling Workstation, reference markers must be placed on
the Bind-Silane-coated plate.
μ
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