Biology Reference
In-Depth Information
Fig. 1. Impact of PMT voltage on spot volumes. The two images show a small gel section scanned with a PMT potential of
450 V ( left ) and 650 V ( right ). At the lower PMT voltage, the volumes of all fi ve spots within the section are quantifi able,
whereas at higher voltage, the signal of one spot runs into saturation. In 3D view, signal saturation manifests itself in a
characteristic peak truncation.
Fig. 2. Impact of pixel size on image resolution. The three images show a small 2D gel section scanned with pixel size
settings between 1,000 and 100 μ m. A pixel size above 500 μ m is not suffi cient to resolve distinct protein spots properly.
saturation is given in Fig. 1 . In case that protein spots are recognizably
affected by signal saturation, the PMT voltage has to be reduced to
enhance the dynamic range of the gel image. The PMT voltage has
to be checked for all three fl uorescence channels to prevent signal
saturation effects.
The pixel size defi nes the resolution of a gel image. A small pixel
size (25-100
Pixel Size
m) results in a high-resolution image and is associ-
ated with an extended scanning time and an increased size of the
image fi le generated. A pixel size of 1,000
μ
m is typically used for
fast pre-scans to estimate whether the PMT voltage has to be
adjusted. 2D imaging is carried out using resolutions around
100
μ
m. As demonstrated in Fig. 2 , this resolution is fully suffi -
cient to perform image analysis. A further increase in resolution
would only increase the size of the generated image but not its
information content.
μ
Apart from all technical considerations, it is of crucial importance
that the generated image fi les can be assigned to the appropriate
laser fl uorescence channel. It is highly recommended to use an
unambiguous fi le-naming scheme (i.e. extending each image fi le
name by a short abbreviation of the associated channel) and to save
image fi les derived from identical gels in one folder (see Note 4).
File Naming
and Documentation
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