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Fig. 1. Selection of relevant cell types. Tumor cells ( a , b ) or bronchial epithelium cells
( c , d ) can be identifi ed in tissue slices after staining with hematoxylin and eosin. Tissue
for collection of cells by microdissection for subsequent proteome analysis (marked in b , d )
should be stained without the use of eosin. The bar represents a distance of 100 μ m .
2.
Prepare three aliquots containing 1,000, 4,000, and 5,000
cells in 50
μ
L of lysis buffer each (see Note 4).
3.
Add 2 nmol TCEP, i.e., 1
L of the 2 mM TCEP solution, to
each sample and incubate for 1 h at 37°C. Label the samples by
incubating them with 4 nmol saturation dye (either Cy3 or
Cy5 can be used, but all samples should be labeled with the
same dye) for 30 min at 37°C in the dark (see Note 5). For
stopping the labeling reaction, add 5
μ
μ
L of 1.4 M DTT and
5
μ
L of Servalyt 2-4 before 2DE.
4.
Subsequent to 2DE (see Subheading 3.5 ) scan the gels as
described elsewhere (see Note 6) and detect the number of
spots using the differential in-gel analysis (DIA) module of
the DeCyder™ software (see Note 7). The optimal number
of cells considered for subsequent analyses is found if the
spot number cannot be signifi cantly increased by applying a
higher number of cells (see Fig. 2 ).
1.
Having established the optimal number of cells for compre-
hensive proteome coverage, one must empirically optimize the
labeling conditions. This will avoid the effects of over- and
under-labeling. Therefore, a so-called “same/same” experi-
ment titrating different dye amounts is performed. For this
3.3. Optimization
of Fluorescence
Dye Labeling
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