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7. Low-fl uorescence glass plates are used to reduce background
in this experiment.
8. Only one plate in each set should be treated with Bind-Silane;
treat the smaller, nonspacer plates if using Ettan DALT 24 cm
gel plates. Bonding allows easier handling of gels during
scanning, protein staining, storage, and importantly, for robotic
spot excision (see Fig.
3
).
9. By increasing PMT voltages, it is possible to increase the over-
all sensitivity of detection while saturating only a few of the
signals from the more abundant proteins. This allows analysis
of lower-abundance proteins that give reasonable signal-to-
noise ratios.
10. In the protocol outlined, we are essentially measuring the
change in R-SH reactivity upon oxidative insult. However, the
method can be adapted to identify proteins whose thiols have
become oxidized by the insult. In this case, the samples are fi rst
alkylated (e.g., with
N
-ethylmaleimide or iodoacetamide) to
block existing R-SH groups, and then reduced (e.g., with DTT
or TCEP) to regenerate R-SH groups from the oxidized forms
prior to ICy dye labeling. Both alkylating and reducing agents
must be removed after each step, either by protein precipita-
tion or by size-exclusion columns.
11. Gels in containers should be placed on a rocking platform for
staining. In addition, containers should be properly sealed to
avoid evaporation.
12. The use of siliconized or low-bind tubes avoids protein or pep-
tide loss due to adsorption on the tube walls and also avoids
contaminants derived from the tube plastic.
13. Make sure the gel pieces are completely dried.
14. It is also possible to search for the ICy dye modifi cations and
mono-isotopic masses of 512.36 Da [ICy3-I+H]
+
and
510.36 Da [ICy5-I + H]
+
should be added to searches as vari-
able modifi cations. It should be noted, however, that we have
had diffi culty observing the modifi cations in digested gel
extracts. We suspect that the poor recovery may be due to the
lowered solubility and ineffi cient extraction of labeled peptides
from gel pieces, since model peptides labeled in solution
performed well in MALDI-TOF experiments.
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