Application of Saturation Labeling in Lung Cancer Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 256

3. Apparatus for vertical SDS-PAGE (System VA Sarstedt,

Nümbrecht, Germany).

4. Gel carrier grooves (homemade).

5. Gel solution: 376 mM Tris-HCl, pH 9.4, 0.03% (v/v)

TEMED, 3.5 mM SDS, 15% (w/v) acrylamide, and 0.2%
(w/v) bisacrylamide: store in aliquots at −20°C.

6. Running buffer: 25 mM Tris base, 192 mM glycine, and

3.5 mM SDS: store at room temperature.

7. 40% (w/w) APS: store in aliquots at −20°C.

8. Protection solution: 376 mM Tris-HCl and pH 9.4, 3.5 mM

SDS: store in aliquots at −20°C.

9. Agarose solution: 1% (w/v) agarose (dissolved in running buf-

fer) and 0.001% (w/v) bromophenol blue.

3. Methods

Manual microdissection as well as laser capture microdissection is a
time-consuming process. Depending on the cell type, to harvest
the collection of 1,000 cells (~1 g extracted protein), approxi-

mately 1-2 h is needed. Therefore, high sensitive protein detection
using protein saturation labeling is a method of choice for quanti-
tative protein analysis ( 5 ). Differences between minimal labeling
and saturation labeling prior to two-dimensional electrophoresis
(2DE) are listed below:

μ

If maleimide dyes are used instead of NHS ester dyes, prior to

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the covalent attachment of the dye via a thioether linkage, the
proteins must be reduced using TCEP. The concept is to label
all cysteine residues of a protein sample; therefore, proteins
without a cysteine residue cannot be detected.
The saturation labeling reaction is performed at pH 8 and

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37°C (minimal labeling: pH 8.5 at 4°C).
As the amount of cysteine residues might vary from sample to

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sample, it is mandatory to determine the optimum amount of
TCEP and dye for each type of sample. A low amount of TCEP
and dye can result in an incomplete labeling of proteins leading
to vertical streaks and nonoptimal overlay in 2D images. If the
amount of TCEP and dye is too high, unspecifi c labeling might
occur. Additional labeling of lysine groups reduces the proteins'
charge and fi nally might lead to horizontal streaks in 2D images.
Over and under-labeling can be controlled by performing a
“same/same” experiment (see Subheading 3.3 ). In this experi-
ment, same amounts of protein in both channels (Cy3 and
Cy5) are applied, whereas the amount of dye is varied until
optimal labeling conditions are found.