Biology Reference
In-Depth Information
the DMF bottle to trap any water and discard the DMF when
the molecular sieve becomes viscous. Protect the dyes and the
labeled samples from light exposure.
6. It is advantageous to store appropriate volumes of the equili-
bration buffer stock solution (50 mM Tris-HCl (pH 8.8), 6 M
urea, 30% (v/v) glycerol, and 2% (w/v) SDS) at −20°C.
Required volumes of iodoacetamide or DTT should be added
just prior to use.
7. To maintain reproducibility especially for large-format 2D
gels (20 × 26 cm), you have to pay increased attention to the
casting conditions. For consistent results, we strongly recom-
mend to develop a standard protocol defi ning all factors like
polymerization time and temperature, high-quality chemicals,
etc. Alternatively, precast gels are available from different
suppliers (Gelcompany, GE Healthcare, etc.) including options
with low-fl uorescent plastic backing required for sensitive
DIGE profi ling.
8. When gels are scanned directly between glass plates, low-
fl uorescent glass plates should be used for gel casting to achieve
highest sensitivity (see also Note 7). Best scanning results can
be achieved with clean “gel packages”; rinse the plates with
water and wipe them dry using lint-free paper. Reduced
background will signifi cantly improve and facilitate software-
based pattern analysis.
9. For best results, samples should be free of precipitate as suspended
components may coat the active bead surface. Therefore, in
order to clarify the sample prior to ProteoMiner™ treatment, a
centrifugation step (10,000 ×
g
for 10 min) should be included,
thus improving the capture effi ciency of the beads as well as the
reproducibility. Do not use hemolytic samples (red color).
Please note that for the high-capacity columns the protein
concentration has to be above 50 mg/mL which is usually the
case for typical serum or plasma samples.
10. In most publications captured, proteins are eluted using acetic
buffer conditions (recommended by the manufacturer).
Although this elution procedure can easily be combined with
other profi ling techniques like SELDI, its use for 2D electro-
phoresis approaches like DIGE needs accurate pH adjustment
or precipitation of the eluate prior to the IEF step. To circum-
vent this additional treatment step we established an elution
protocol using 2D DIGE lysis buffer, which in our hands works
as well as the acetic elution (see Fig.
1
). Interestingly, our
procedure levels the pH in almost all cases automatically to 8.5
which is exactly the pH needed for the DIGE labeling reaction
making this protocol a very convenient alternative to the acetic
elution. Nevertheless, we recommend checking the pH of
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