Biology Reference
In-Depth Information
In contrast to minimal labeling (Cy2, Cy3, and Cy5 dyes are
●
available), only two dyes (Cy3 and Cy5) are available for satu-
ration labeling. Therefore, the experimental design is different:
the internal standard is labeled with Cy3, whereas the sample is
labeled using Cy5, and no color swapping is performed. The
use of the internal standard minimizes variations introduced by
the system and corrects for quenching effects of highly labeled
proteins (
6
) ensuring highly accurate quantifi cation of relative
protein abundance.
For protein identifi cation after in-gel digestion, protein amounts
●
of a saturation labeling experiment are too low, a reference pro-
teome on a preparative gel has to be applied with a protein spot
matching rate of >90% for the analyzed sample. As a source for
such a reference proteome sample, tissue resection or cell culture
cells can be considered, but the selection for the optimal refer-
ence proteome has to be determined empirically. Once a sample
for the generation of a reference proteome has been selected,
this reference proteome is applied as the internal standard for
the analytical gels. For preparative gels (e.g., 400
g protein), a
higher concentrated Cy3 dye (20 mM) is used. It should be
kept in mind that the reference proteome concentration should
be >2
μ
L, thus not exceeding the maximum loading vol-
ume at the isoelectric focusing (IEF) step.
μ
g/
μ
1. Harvest relevant tissue specimens (neoplastic and non-neoplastic
peritumoral samples) (see Fig.
1
), freeze resections on liquid
nitrogen and store at −80°C (see Note 1). For histological clas-
sifi cation of tumor tissue, prepare frozen 5-
3.1. Manual
Microdissection
m sections from
tissue blocks using a cryostat. Place sections briefl y in 96%
ethanol and stain with hematoxylin and eosin for immunohis-
tological inspection.
μ
2.
For microdissection from subsequent tissue slides, prepare fro-
zen 10-
m sections from tissue blocks containing the required
tumor grades or non-neoplastic tissue. Place them briefl y in ice
cold 96% ethanol and only stain with hematoxylin (see Note 2).
μ
3.
Under microscopic observation, harvest the required number
of cells (see Note 3) using a sterile injection needle directly
into 50
μ
L of lysis buffer (4°C).
4.
Sonicate (6 × 10-s pulses on ice) after each collection step and
fi nally centrifuge the lysate for 5 min at 12,000 ×
g
and store
the supernatant at −80°C.
3.2. Determination
of the Minimal Number
of Microdissected Cells
1.
Because microdissection is time-consuming and the number of
available sample material is limited, it is necessary to fi nd the
minimal number of cells suffi cient for a differential proteome
analysis. Therefore, a pool of approximately 10,000 microdis-
sected cells is required to determine the optimal number of
spots by a dilution experiment.
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