Biology Reference
In-Depth Information
Table 1
Overview of 2D DIGE-based studies with marine (environmental) bacteria from the
author's laboratory
Organism
Research topic
Reference
Marine aerobic heterotrophs
Rhodopirellula baltica
SH1
Evaluation of 2D DIGE
Peripheral/central pathways of carbohydrate
metabolism
Growth rate-dependent regulation of protein
composition
(
30
)
(
40
)
(
41
)
Phaeobacter gallaeciensis DSM 17395
Central metabolism
(
47
)
Anaerobic degradation specialist
Aromatoleum aromaticum
EbN1
Anaerobic degradation of ethylbenzene
and toluene
Catabolic network across >20 substrate
conditions
Anaerobic degradation of
p
-ethylphenol
Solvent stress response
(
54
)
(
56
)
(
55
)
(
57
)
simplicity—composition of the internal standard ultimately limits
the number of test states that can be combined in a single working
package. Protein extracts are generated, for example, from 16 distinct
cell pellets, representing four biological replicates for each of the
four different growth conditions: one reference state (R1-R4) and
three test states (A1-A4, B1-B4, and C1-C4). For each labeling
reaction, 50
g of protein extract is labeled with 200 pmol cyanine
dye. The type of cyanine dye applied depends on the experimental
designation of the protein extract(s): reference state (R; Cy5 dye),
test states (A, B, C; Cy3 dye), or pooled internal standard (IS; Cy2
dye). Each biological replicate of the reference state is labeled at
threefold amount. Each biological replicate of the test states is
labeled at onefold amount. The internal standard contains all sam-
ples (4× reference state, 12× test states) included in the working
package and is labeled at 12-fold amount.
μ
The labeled protein extracts are mixed according to the experi-
mental design to yield 12 separate mixtures, each containing equal
amounts of the reference state, one of the three test states, and the
internal standard.
2.1.2. Mixtures of Labeling
Preparation
Separation of protein mixtures is performed by 2DE using immo-
bilized pH gradients essentially according to the procedures
developed by Görg and coworkers (
32
).
2.1.3. Two-Dimensional
Gel Electrophoresis
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