Biology Reference
In-Depth Information
Following 2DE, gels are imaged with a three-mode fl uorescence
scanner (Typhoon 9400), yielding three independent CyDye-specifi c
images from one gel. The scanner is operated at a resolution of
100
2.1.4. Image Acquisition
m, and gel images are generated with optimal maximum
pixel intensities of 50,000-80,000.
μ
Prior to analyses with the DeCyder software, gel images are cropped
to exclude nonessential parts. In our laboratory, a spot detection
limit of 3,000 spots is used. The spot detection parameters are set
to slope >1, area <200, height <190, and volume <6,000. The gel
with the best resolution and highest number of spots is selected as
master gel. For intergel comparison in the biological variation
analysis (BVA) module, 10-20 common landmarks are set on the
master gel and subsequently across all other gels. Proteins with
changed abundances are manually controlled and have to fulfi ll
the following criteria: average ratio of <|2-2.5|, ANOVA
P
-value
of <0.05,
t
-test value of <10
−4
, and matched in at least 75% of all
analyzed gels.
2.1.5. Image Analysis
2.2. Ongoing
Developments
Current methodological work in our group on 2D DIGE is con-
cerned with evaluating the infl uence of biological vs. technical
variation on fold-changes of protein abundance (average ratio)
(
33
). Moreover, we are interested in defi ning the lowest, while still
robust thresholds of signifi cance, i.e., what is the minimal protein
abundance fold-change that represents a biological meaningful
response. In our experience, catabolic proteins display strong
abundance fold-changes in response to the respective substrate(s).
In such cases, a conservative threshold (>|2.5|) is reasonable since
it allows avoiding too many false positives. However, in case of cel-
lular or global regulatory processes, lower fold-changes may actually
be of biological signifi cance. Finally, the threshold of signifi cance
may have to be determined separately for each model organism.
3.
Rhodopirellula
baltica
SH1:
Heterotrophic
Marine
Planctomycete
Planctomycetes are widespread in nature (freshwater, marine,
soils); display unusual morphologies, such as rosettes, stalks, and
intracellular compartmentalization (
34, 35
); and belong to a dis-
tinct phylogenetic superphylum (
36
). The marine, aerobic, and
heterotrophic
Rhodopirellula baltica
SH1 (
37
) was the fi rst planc-
tomycete to be genome-sequenced (
38
) and investigated on the
proteome level, including establishment of the fi rst 2D DIGE
master gel for planctomycetes (
39, 40
). The presumptive lifestyle
of
R
.
baltica
SH1 in the marine water column involves energy
3.1. Background
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