Biology Reference
In-Depth Information
weight and will be more of an issue for the lower molecular weight
proteins, but this procedure should be performed as standard prac-
tice. Spots of interest can now be matched to the pick gel image by
using the analysis software. The pick gel can be run as a separate
preparative gel on its own, or extra unlabeled protein (made up as
for the internal standard) can be added equally to all of the analyti-
cal gels such that each gel is then a potential pick gel.
3.7. Spot Processing
and Identifi cation
The excised protein spot can be enzymatically digested (usually
with trypsin), and the resulting peptides can be analyzed with a
mass spectrometer. The most commonly used techniques include
matrix-assisted laser desorption/ionization (MALDI) or electro-
spray ionization (ESI) mass spectrometry (MS). Once a protein
has been identifi ed by MS, it can be very useful to verify its identity.
This can be achieved by Western blotting if an antibody is available
against the target of interest. Western blotting combined with a
2D gel can be a very powerful approach since an SDS-PAGE gel
alone will not detect the posttranslational modifi cations that result
in different charge forms of the same protein being present. If
blotting an actual 2D DIGE gel from an experiment, it is impor-
tant that the reporter molecule should not interfere with the signal
from the CyDyes such that the antibody can be linked to an enzyme
(such as horse radish peroxidase, HRP) for chemiluminescent
detection or the antibody can be linked to an infrared reporter
molecule. It must be remembered that the total protein from con-
trol, treated, and internal standard will now be detected, so this
approach is more useful for confi rmation of location and identifi ca-
tion. However, if using a two-dye system (see Note 6) for the
DIGE experiment (e.g., Cy 3 and Cy5), then the third dye (Cy2)
could be used as a reporter molecule on the primary or secondary
antibody.
4. Notes
1. Reproducibility of spot patterns can be facilitated by the use of
precast gels for both the fi rst and second dimension.
2. To facilitate accurate spot picking, it is strongly recommended
to immobilize the gel to prevent swelling or shrinking during
the staining procedure. This can be achieved by treating one of
the two low fl uorescent glass plates (one pair of plates is used
per gel) with Bind-Silane. Another approach is to use a low
fl uorescent plastic-backed gel.
Reference markers can be attached to the support surface
prior to gel casting or imaging to enable more accurate spot
picking with robotic instrumentation—these markers will serve
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