Biology Reference
In-Depth Information
2. Materials
For the imaging methods described below, the preparation of a
2D DIGE gel is needed. Most of the examples are based on
employing consumables and equipment from GE Healthcare
(Freiburg, Germany), including CyDyes™, Typhoon™ laser scanner
and DeCyder™ software.
3. Methods
3.1. Preliminary
Considerations
The prerequisite for successful gel imaging and for all subsequent
spot detection and quantifi cation approaches is the use of high-
quality 2D polyacrylamide gels.
The easiest but more expensive way to obtain suitable gels is to
purchase commercially available pre-cast gels that comply with the
standards of good manufacturing practice. There are two concepts
of gels available: pre-cast gels in gel cassettes and fi lm-backed.
Before deciding on one of these concepts, its compatibility with
the existing image acquisition hardware has to be evaluated. For
example, in case of deciding on a fi lm-backed gel in combination
with a laser fl uorescence scanner, the gel backing foil has to show
low fl uorescence properties and should be translucent for light in
the near UV spectral range. Although pre-cast gels are commer-
cially available, many users still prefer lab-cast gels for economic
reasons. In case of deciding on casting gels in one's own lab, the
whole casting procedure should be part of a very precise and well-
established multifl uorescence 2D gel standard operating protocol
(SOP). The acrylamide monomer solution has to be made of very
high-quality chemicals and should be degassed prior to gel casting.
In order to obtain sets of lab-cast gels with similar physical and
chemical properties, it is necessary to use a multi-gel caster. In case
of using a fl uorescence scanner equipped with confocal laser optics
for image acquisition, the gel casting cassettes should be made of
low-fl uorescent glass, allowing scanning directly between the glass
plates. During gel casting, special attention has to be paid to gel
homogeneity and a straight gel surface: It is recommended to
pre-cool the acrylamide monomeric solution with a relatively low
concentration of ammonium persulfate and to overlay the gels by
spraying 0.1% sodium dodecyl sulphate into the gel casting cassettes
before the monomer solution starts to polymerize. After polymer-
ization, the gels should be overlayed with 2D running buffer and
stored for a reasonable period of time (about 4 days) at 4°C to
ensure complete polymerization. The storage time should be
defi ned and controlled by an SOP.
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