Combination of Highly Efficient Hexapeptide Ligand Library-Based Sample Preparation with 2D DIGE for the Analysis of the Hidden Human Serum/Plasma Proteome - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 174

Fig. 2. DIGE analysis of fractionated serum samples to test column-to-column reproducibility. A reference serum sample
was divided into three equal aliquots (1 mL), and each was separated on a different ProteoMiner™ column. Captured
proteins were eluted using 2D DIGE lysis buffer; proteins were labeled with CyDyes and separated in the fi rst dimension by
IEF (pH 4-7) and in the second dimension on a 12%-SDS-PAGE gel. Protein patterns were visualized by laser scanning,
and image analysis of the resulting nine images (three different columns crosswise labeled with Cy3 and Cy5 and internal
standard labeled with Cy2) was performed using Proteomweaver™ software. Comparing the spot profi les from the
three different columns reveals high reproducibility between the different replicates with CVs less than 20%. Blue dots in
the scatter plot indicate intensity of detected protein spots in each group.

3.2. Hexapeptide
Library Sample
Preparation

1. Serum samples were thawed at RT (be sure that samples are
fully thawed) and centrifuged for 10 min at 10,000 × g at 4°C
to remove sample particulates. Only the supernatant was used
for the subsequent steps (see Note 9).
2. Top and bottom caps from the ProteoMiner™ columns were
removed and columns centrifuged for 2 min at 1,000 × g . The
fl ow-through was discarded. All subsequent centrifugations
were for 2 min at 1,000 × g .
3. After addition of 1 mL of deionized water and replacement of
the caps, the columns were rotated end-to-end for 5 min. Caps
were removed, columns centrifuged again, and the fl ow-through
was discarded. The centrifugation step was repeated once.
4. After addition of 1 mL of wash buffer and replacement of the
caps, the columns were rotated end-to-end for 5 min. Caps
were removed, columns centrifuged, and the fl ow-through was
discarded. The centrifugation step was repeated once.
5. After addition of 1 mL of sample (serum) and replacement of
the caps, the columns were rotated end-to-end for 2 h at room
temperature. Caps were removed, columns centrifuged, and
the fl ow-through was discarded. The centrifugation step was
repeated once.
6. After addition of 1 mL of wash buffer and replacement of the
caps, the columns were rotated end-to-end for 5 min. Caps
were removed, columns centrifuged, and the fl ow-through was
discarded. The centrifugation step was repeated once and the
entire wash step was repeated 2-3 times.