Biology Reference
In-Depth Information
The aliquots can be stored at −80°C for >12 months without
appreciable loss of sensitivity. When required, simply add the
sample directly to the tube of dye.
2. Set up buffer A and buffer B as the mobile phases. Purge both
lines at a fl ow rate of 1 mL/min for 10 min with the column off-
line. Set up the chromatography timetable (see Subheading
3.1
,
step 2) and perform two blank runs with 100-mL injections of
buffer A with the column off-line. Attach the column and
equilibrate with buffer A for 4 min at 1 mL/min. Place the
MARS column online and perform a blank injection of 100 mL
of buffer A at 0.5 mL/min.
3. Additional acetone washing does not reduce the conductivity
of the samples and thus is unnecessary, potentially resulting in
sample loss.
4. If samples are frozen at this stage, all subsequent processes
(i.e., buffer exchange, DIGE labeling, and 2DE) should be
performed without refreezing the sample. Repeat freeze-thaw
cycles should be avoided.
5. It is important not to centrifuge at colder temperatures, other-
wise the urea will precipitate and concentrate in the Vivaspin
device.
6. This is not the standard dye/protein ratio recommended by
the manufacturer of the CyDyes (GE Healthcare). In our expe-
rience, however, the effi ciency of labeling at 200 pmol/100 mg
of protein is comparable to the recommended 400 pmol/50 mg.
The economic benefi ts of reducing the dye requirement are
signifi cant. Furthermore, we have found that a total 300 mg
(100 mg contributed from each labeling reaction) is the maxi-
mum amount of protein that can be run on a large-format gel
without affecting spot resolution, yet still provides enough
material for subsequent MS analysis. This negates the require-
ment for a preparative gel(s) in the experiment as spots are
excised directly from the analytical gels.
7. It is important to use reverse osmosis rather than ultrapure
water for soaking the paper wicks as ultrapure water is not suf-
fi ciently conductive.
8. Keep the coversheet that is used to protect the precast gels
prior to electrophoresis. It is useful when it comes to storing
the gels in the freezer.
9. It has been found that the gels can be safely thawed at room
temperature and rescanned after several months of storage at
−80°C (possibly indefi nitely) without signifi cant diffusion of
the protein spots or damage to the gel or support fi lm.
10. The inclusion of 10% acetonitrile in the digest solution ensures
the trypsin has maximum activity and helps to maintain the
solubility of hydrophobic peptides.
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