2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 94

of which may require special detergents, whose compatibility has to
be assured by separate experiments. In saturation DIGE, the user
typically handles very limited sample amounts, leading to additional
challenges with respect to reproducibility. Consequently, all steps
which are not absolutely essential should be avoided to facilitate the
highest possible degree of reproducibility. It is more effective to
consciously disregard some protein analytes (e.g., hardly soluble
proteins) in favor of a reproducible procedure than to end up with
lysates differing in their protein composition within biological rep-
licates. While the former will simply lead to a lack of information
about distinct individual proteins, the latter might lead to a com-
plete failure of the experiment as a consequence of abundance dif-
ferences introduced by the procedure. Here, we describe a sample
preparation procedure based on the buffer recommended by GE
Healthcare. Note that this buffer must not contain a disulfi de-
reducing agent (see Note 1).

1. Carefully wash your tissue or cells at least 4 times, e.g., with
PBS (phosphate buffered saline), at 4°C to remove blood and
any contaminants contained in the culture media. Carryover of
serum or media proteins can have disastrous consequences and
limit the scientifi c outcome of the experiment (see Note 2).
2. Centrifuge after each washing step for 10 min at 600 × g , 4°C,
and remove the supernatant carefully and completely. Even
traces of remaining wash buffer may signifi cantly reduce the
concentration of chaotropic agents in the lysis buffer and affect
extraction and solubilization of proteins. After the last centrif-
ugation step, remove even the last microliter of remaining wash
buffer by aspiration with a very thin glass capillary or a pipette
tip used for ultrathin gel loading.
3. Add lysis buffer to end up with a total protein concentration of
>0.55 μ g/ μ L and <5.0 μ g/ μ L for analytical gels (see Notes 3

and 4).
4. Check if the protein amount to be analyzed per 2D gel is
contained in a volume which can be readily applied to your
isoelectric focusing device (see Note 4).
5. Apply a lysis procedure (e.g., sonication, freeze thaw) suitable
for the sample analyzed.
6. Check for solubilization after centrifugation at 10,000 × g for
5 min at 4°C. Ideally, no pellet at all is visible after completion
of the lysis procedure (see Note 5).
7. Transfer the supernatant to a fresh microfuge tube. Check if
the pH is still in the range of 7.8-8.2 (see Note 6).
8. If the pH is below 7.8, titrate to pH 8.0 by stepwise addition
of lysis buffer adjusted to pH 9.5 instead of 8.0.