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

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3.

Thaw the pellet at room temperature and resuspend it as small

particles in 700

μ

L of 100% ice-cold acetone using the end

part of a 200-

L tip or long-nose tip and repeated aspiration

and dispensing. Centrifuge at 20,000 × g for 10 min, and dis-

card the supernatant. Resuspend again in 700

μ

L of 100% ice-

cold acetone, centrifuge at 20,000 × g for 10 min, and discard

the supernatant. Move the pellet against the tube side to easily

dissolve it in the lysis buffer using the end part of a tip and dry

the pellet at room temperature for 5 min. Add an adequate

lysis buffer volume (usually 100-150

μ

L), vortex gently to

prevent the creation of any bubble for 5 min, detach the non-

dissolved pellet from the tube wall using a tip, and then vortex

again as described above. Centrifuge at 20,000 × g for 20 min

at 4°C, recover the supernatant, and adjust the protein solu-

tion to pH 8.0-9.0 with 1N NaOH, as assessed with a pH

indicator strip. Measure the protein concentration and adjust

1,000

μ

g of each sample to 5

μ

g/

μ

L concentration for CyDye

labeling (see Note 5).

1.

Prepare the pooled standard (25

μ

g each of normal and HCC,

3.2. CyDye Minimal

Labeling and Protein

Separation by 2DE

pooled into one 50

μ

g total sample), normal (50

μ

g), and

HCC (50

g) samples as shown in Table 1 (see Note 6). Add

400 pmol of the appropriate dye (Cy2, Cy3, or Cy5) to each

sample and vortex, then incubate on ice in the dark for 30 min

μ

Table 1

Experimental design for 2D DIGE using reciprocal labeling,

two replicates, and a pooled internal standard

Gel no.

pH range

Cy2

Cy3

Cy5

1

3.5-4.5

Pooled standard (normal + HCC) Normal HCC

2

4.0-5.0

Pooled standard (normal + HCC) Normal HCC

3

4.5-5.5

Pooled standard (normal + HCC) Normal HCC

4

5.0-6.0

Pooled standard (normal + HCC) Normal HCC

5

5.5-6.7

Pooled standard (normal + HCC) Normal HCC

6

7.0-11.0 Pooled standard (normal + HCC) Normal HCC

7

3.5-4.5