Biology Reference
In-Depth Information
which permits scanning the gels right after the electrophoretic run
without any additional staining step. Furthermore, in this way up to
three different samples may be compared on one gel (
2, 3 ).
In its basic methods, animal proteomics is not different from
other proteomic varieties, relying on the same principles and apply-
ing the same methods. In most cases, the 2 DE protocol has to be
adapted to the sample type rather than to the species. “Species-
specifi c” problems may derive from sample collection or pretreat-
ment (most commercial kits are designed for human sample
material), properties of homologous or species-specifi c proteins,
and/or the incompleteness of animal protein databases (for subse-
quent identifi cation by mass spectrometric analysis).
The way to perform 2 DE described here is—apart from apply-
ing modern DIGE technology—a bit “old-fashioned”, meaning
that it does not rely on commercial precast gels. It is thus more
time-consuming, includes more manual steps, and therefore requires
some experience. But, on the other hand, it is more fl exible, as
every detail, particularly pH gradient and size of the IPG strip, but
also additives and second dimensional running conditions, can be
adapted to one's own needs and thus may include specifi cations or
varieties not commercially offered. The system has served quite well
over years and is rather robust, allowing application to all kinds of
samples, among them different body fl uids, cells, and tissue prepa-
rations, mainly of animal origin (e.g., (
4-6 )). The main sections
describe 2D DIGE of serum, with an optional step for MS-compatible
silver staining. Tips and comments on other sample material, gel
size, or separation systems are also provided.
2. Materials
All solutions are prepared with ultrapure water (corresponding to
Milli Q quality) and chemicals of p.a. quality.
2.1. Sample
Preparation/DIGE
Labeling
1. DIGE labeling buffer: 8 M urea, 4% CHAPS, 30 mM Tris-HCl,
pH 8.5; the pH is checked with pH indicator paper (see Note 1).
The buffer may be stored in appropriate aliquots at −20°C.
2.
Dyes: CyDye DIGE fl uor Cy2, Cy3, Cy5 minimal dyes (GE
Healthcare, Munich, Germany); stock solutions for the dyes are
prepared in anhydrous dimethylformamide (DMF; see Note 2).
The tube with the solid dye is briefl y spun, and DMF is added
to achieve a 1-mM dye solution (e.g., 5 nmol dye/5 mL), mixed
vigorously, and centrifuged to collect the solution at the bot-
tom of the tube. As a working solution, a 1:2.5-dilution either
with DMF or with DIGE labeling buffer may be used.
3.
Stop solution: 10 mM lysine, freshly prepared.
Search WWH ::




Custom Search