Biology Reference
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Fig. 3. Optimization of saturation labeling. Protein extracted from 5,000 cells was labeled
with 4 nmol (
a
,
b
) and 8 nmol (
c
,
d
) dye, respectively: Cy3 (
a
,
c
) and Cy5 (
b
,
d
). Samples
labeled with the same amount of dye were co-separated by 2DE. Whereas labeling with
4 nmol dye resulted in a good overlap of spot maps (
a
,
b
), the higher amount of dye
resulted in unspecifi c additional attachment of dye to proteins. For the latter, some
proteins shift in horizontal direction (
arrows
in
c
,
d
), probably because of unspecifi c
dye attachment to lysine residues, which subsequently lost their charge.
identification. A reference proteome could therefore be
prepared from tumor cell lines or solid tissues (e.g., sections
of tumor tissue grade 2 and grade 3).
3.
Prepare 25-
m-thick sections of tumor tissue and epithelial
tissue (esophagus) using a cryostat. Homogenize ~100 mg of
the tissue on ice in 240
μ
L of lysis buffer using a hand homog-
enizer and sonicate the sample 6 times for 10 s on ice.
μ
4.
To remove insoluble debris centrifuge the homogenate for
5 min at 12,000 ×
g
.
5.
Perform a Bradford assay to determine the protein concentra-
tion of the samples.
6.
Label 5
g of each lysate with 4 nmol Cy3 according to the
standard labeling protocol (see Subheading
3.2
, step 3).
μ
7.
Label aliquots of 5,000 microdissected cells with 4 nmol Cy5.
8.
Pairwise mix the labeled tissue lysates with labeled cells and
process by 2DE.
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