Differential Gel-Based Proteomic Approach for Cancer Biomarker Discovery Using Human Plasma - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 229

(see Note 7). Quench by adding 1 L of 10 mM lysine and

incubate on ice for 10 min. Mix the three samples (150

μ

g)
together, and add an equal volume of 2× sample buffer to a
fi nal volume of 450

μ

L. For each preparative gel, mix 1 mg of
unlabeled pooled standard proteins and sample buffer to a fi nal
volume of 450

μ

μ L.

2. Mix 9 L of the

protein solution and incubate for 30 min at room temperature.
Rehydrate Immobiline 24-cm Dry Strips of the six pH ranges
with protein solution in the strip holder for 16 h at room tem-
perature. Perform fi rst-dimension isoelectric focusing (IEF)
using the MultiPhor II electrophoresis system at 20°C with the
following conditions: step 1: 100 V for 4 h, step 2: 300 V for
2 h, step 3: 600 V for 1 h, step 4: 1,000 V for 1 h, step 5:
2,000 V for 1 h, step 6: 3,500 V for 29 h (see Note 8).

μ L of IPG buffer for each pH range into 450 μ

3. Before the end of the IEF process, prepare all 9-16% 2-D gels,

using 12 LF glass plates for the 2D DIGE gels and six general
glass plates for the preparative gels.

4. After IEF, transfer the strips into capped glass tubes and soak

the strip gels in equilibration buffer containing 5 mM TBP for
25 min (see Note 9). Apply the strips onto the precast 9-16%
2-D gels. Perform electrophoresis with an Ettan DALTtwelve
electrophoresis system using the following electrophoresis
conditions at 20°C: step 1: 2.5W/gel for 30 min, step 2: 10W/
gel for 3 h, step 3: 16W/gel for 4 h.
5. Scan the gels containing the DIGE-labeled proteins using a
Typhoon 9400 Imager ® set for the excitation/emission wave-
lengths of each DIGE fl uor; Cy2 (488/520 nm), Cy3
(532/580 nm), and Cy5 (633/670 nm) (see Note 10). Crop
and save the area of interest using ImageQuant V2005 software.

6. Fix each preparative gel in fi xing solution for 2 h. Stain with

Coomassie Brilliant Blue G-250 staining solution for 6 h, and
destain by washing with distilled water at least three times.
Scan each gel, and then pack each one in a clean vinyl bag with
water, and store at 4°C.

Figure 1 shows typical 2-D gel spot patterns of whole plasma
(a, b) and plasma depleted of High-abundance protein (HAPs)

(c, d), respectively. In the image of whole plasma, over 90% of
spots contain mainly albumin, IgG heavy and light chain, alpha-
1-antitrypsin, IgA, transferrin, haptoglobin, fi brinogen, apoli-
poprotein A-1, and alpha-1-acid glycoprotein as HAPs. Therefore,
differently expressed targets may be included in less than 10% of
all spots detected and are likely masked by HAPs. The tools for
HAP depletion are commercially available (e.g., Qproteome
Albumin/IgG Depletion Kit, QIAGEN; MARS, Agilent
Technologies; Seppro ® MIXED12-LC20 column, GenWay