2D DIGE Analysis of Serum After Fractionation by ProteoMiner Beads - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 185

Table 1
Experimental design for CyDye™ labeling of four serum samples with the incorporation
of a pooled sample internal standard

Cy2 (internal standard) Cy3 Cy5

Gel 1 50 mg Pool 50 mg of Serum 1 50 mg of Serum 2

Gel 2 50 mg Pool 50 mg of Serum 3 50 mg of Serum 4

Gel 3 50 mg Pool 50 mg of Serum 2 50 mg of Serum 3

Gel 4 50 mg Pool 50 mg of Serum 4 50 mg of Serum 1

Gel 5 50 mg Pool 50 mg of Serum 1 50 mg of Serum 4

Gel 6 50 mg Pool 50 mg of Serum 3 50 mg of Serum 2

Total 300 mg (from a pool of

4 × 100 mg, each from
serum 1 to 4)

included to reduce bias in fl uorescence or labeling from the different
CyDye™. Triplicate gels are conducted to increase the statistical
confi dence of the experiment.

3.2. Fractionation
of Sera Using
ProteoMiner™

All steps in this section are conducted at room temperature.
Incubation and washes on the rotary shaker are all conducted
at 10 rpm.

3.2.1. Column Preparation

1. Remove the top cap of the spin column and snap off the bottom
cap prior to use. Invert this bottom cap and subsequently use
it as a plug for the remaining part of the experiment.
2. Place the spin column in a capless collection tube and centrifuge
at 1,000 × g for 1 min to remove the storage solution. Discard
the storage solution.
3. Replace the bottom cap and add 200 mL of wash buffer to the
beads. Replace the top cap and gently tap the column to mix.
4. Incubate the mixture in the spin column on a rotary shaker for
5 min. Place the spin column into a secondary tube that can fi t
into the rotary shaker if necessary.
5. Remove the top cap and then the bottom cap (see Note 3),
place the spin column back to the same capless collection
tube and replace the top cap. Centrifuge at 1,000 × g for 1 min.
Discard the solution.
6. Repeat steps 3-5.
7. Replace the bottom cap on spin column. The column now
contains 20 mL of settled bead volume that is ready for sample
binding.