Biology Reference
In-Depth Information
Conditions for the fl uorescent scanning of the gels are fi rst
optimised quickly at low resolution, then all of the gels are scanned
under ideal conditions at high resolution. Before scanning, gels are
kept in the separation unit maintained at the sub-ambient tempera-
ture used for electrophoresis:
1. Turn on the Typhoon scanner 30 min before you intend to
use it.
2. Clean the platen and position the gel alignment guide in place.
3. Two gels can be scanned at the same time. Gels are positioned
with the top opening towards the left and the smaller of the
two glass plates in contact with the platen. See the manufac-
turer's manual for details and typical scanner settings.
4. To optimise the conditions, scan one of the gels at low resolution,
typically using a pixel size of 500 mm. Review this scan using
ImageQuant software, and quantify the intensity as denoted by
“Max. Val(pos)” (see Fig.
2
). “Max. Val(pos)” must be less than
100,000 to avoid saturation. Optimum value for this parameter
is in the range 50,000-80,000. However, the PMT settings
for the individual channels must be optimised so that broadly
similar intensities are obtained for each channel.
5. Once the settings have been optimised, scan each gel in turn at
high resolution, typically using a pixel size of 100 mm.
6. Store scanned gels damp at 4°C in plastic bags until ready to
proceed with the visualisation and spot picking, which should
commence as soon as possible after image analysis in order to
reduce further protein diffusion and contamination.
There are several 2D image analysis software packages that can be
used to analyse the images from DIGE experiments and identify
proteins that are either up- or down-regulated. We have found
Progenesis SameSpots to be fairly straightforward to use, and as it
has plenty of on-screen help, only the major steps will be covered
in this protocol.
7. Open the programme and load the gel images for DIGE
analysis. The software permits cropping, fl ipping and rotating
of images, and carries out several checks on the quality of
the images before proceeding further. Images are organised
according to the gels they originated from.
8. Select a reference image. All of the other images will then be
aligned to this image, so it is advisable to pick as reference an
image that is representative and free from streaking and distor-
tion. Mask any area that is to be excluded from the analysis, for
instance the very bottom of the gel that includes the dye front.
9. Align the images to the reference image. Each internal standard
image (Cy2) is matched to the Cy2 image of the reference gel;
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