Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 276

5. 18% heavy acrylamide solution: 270 mL of 30% (w/v)

acrylamide, 113 mL of 1.5 M Tris-HCl, pH 8.8, 30 mL of
water, 31 mL of glycerol, 4.5 mL of 10% (w/v) SDS, 2.3 mL
of 10% APS, 0.14 mL of 10% TEMED.

6. Displacing solution: 0.375 M Tris-HCl, pH 8.8, 50% (v/v)

glycerol, trace amount of bromophenol blue (Bio-Rad, Cat
no: 161-0404).

7. Gel storage solution: 0.38 M Tris-HCl, pH 8.8.

8. TG-SDS running buffer (Amresco, Cat no: 0783-42).

9. Ettan DALT twelve separation unit (GE Healthcare, Cat no:

80-6466-27).
10. DALT cassette rack (GE Healthcare, Cat no: 80-6467-98).
11. DALT blank cassette insert (GE Healthcare, Cat no:
80-6467-03).

2.5. Gel Scanning,
Image Acquisition, and
Protein Quantifi cation

1. Typhoon 9410 (GE Healthcare, Cat no: 63-0055-80).

2. Kimtech Science Kimwipes (Kimberly-Clark Professional, Cat

no: 34120).

3. DeCyder 2D 7.2 Software package (GE Healthcare, Cat no:

28-9757-78).

3. Methods

Four individual secretome samples (designated a-d) are prepared
from each of the MDCK, 21D1, and 21D1 + TGF-

3.1. Cell Culture and
Secretome Preparation β cell lines for

DIGE analysis:

1. Culture MDCK, 21D1, and 21D1 + TFG- cells in DMEM

using a T-175 culture fl ask containing 10% (v/v) FCS, at 37°C
in 10% CO 2, as described in ref. 17 .

β

2. Passage upon reaching 80% confl uence with Trypsin/EDTA.

3. Seed cells into ten 150-mm tissue culture dishes and culture

until 70% confl uent.

4. Gently wash each dish with 10 mL of DMEM medium (see

Note 1).

5. Repeat step 4 twice.

6. Add 15 mL of DMEM to each dish, and culture for 24 h (see

Note 2).

7. Collect the conditioned medium (CM) from each dish and

pool (see Note 3).

8. Centrifuge the CM at 480 × g at 4°C for 5 min to pellet intact

cells, and transfer the supernatant to a new tube.