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then mixed and subjected to 2DE on a single gel, and protein spot
patterns are visualized by alternately illuminating the gel with the
excitation wavelengths of each of the dyes. The resulting images
are then overlaid and analyzed with software such as DeCyder (GE
Healthcare) to determine proteins that are differentially expressed
between samples. Following analysis, gel spots are excised and
subjected to MS for protein identifi cation.
In this chapter, we describe the experimental workfl ow to
analyze changes in the secretome of Madin Darby canine kidney
(MDCK) cells undergoing oncogenic Ras (21D1 cells), and Ras/
TGF-
-mediated EMT. In order to do so, serum-free culture
conditions were optimized and established, and a secretome
purifi cation methodology developed. Secretome samples from all
three cell lines/conditions were obtained and subjected to DIGE
analysis to reveal proteins that are differentially expressed during
EMT (see Fig.
2
). Following quantitative analysis, MS sequencing
of gel spots of interest revealed that proteins downregulated were
predominantly involved in cell-cell and cell-matrix adhesion, while
upregulated proteins were proteases and factors that promote cell
motility (
36
).
β
2. Materials
2.1. Cell Culture and
Secretome Preparation
1.
Dulbecco's Modifi ed Eagle Medium (DMEM) (Invitrogen,
Cat no: 11995065).
2.
Human transforming growth factor beta (TGF-
β
) (BD
Biosciences, Cat no: 354039).
3.
T-175 Tissue culture fl asks (BD Falcon, Cat no: 353112).
4.
Fetal calf serum (FCS) (Invitrogen, Cat no: 10101-145).
5.
Trypsin/EDTA (Invitrogen, Cat no: R-001-100).
6.
150-mm Tissue culture dishes (BD Falcon, Cat no: 353025).
7.
Complete EDTA-free protease inhibitor cocktail tablets
(Roche, Cat no: 04693124001).
8.
0.1-
m Supor Membrane VacuCap 60 Filter Unit (Pall, Cat
no: 4631).
μ
9.
3K NMWL Amicon Ultra Centrifugal Filter Device (Millipore,
Cat no: UFC900324).
10. 2D Clean-Up Kit (GE Healthcare, Cat no: 80-6484-51).
11. 2DE sample buffer: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS,
20 mM Tris-HCl, pH 8.5.
12. 2D Quant Kit (GE Healthcare, Cat no: 80-6483-56).
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