Biology Reference
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many of the proteins of lower abundance. For example, serum or
plasma is dominated by approximately 20 high-abundance proteins
with concentrations as high as 20-50 mg/mL for serum albumin,
as compared to concentrations as low as mg/mL or pg/mL for some
known biomarkers (
1
). Presently, a number of prefractionation
methods and devices have been developed with the view to reduce
the abundance of these proteins. These include immunoaffi nity-
based methods that are specifi cally designed to remove serum
albumin and in which the separation media are packed either in
spin cartridges or liquid chromatography (LC) columns. However,
there are several recent reports suggesting that potentially interesting
protein candidates may be lost with the removal of albumin via this
approach (
2-7
). ProteoMiner™ or Equalizer beads can be used
as an alternative to address the problem of wide dynamic range of
the proteins in serum and to overcome the loss of proteins by the
removal of “total” albumin via immunodepletion methods. The
ProteoMiner™ technology uses a combinatorial library of hexa-
peptides bound to beads. Due to the tremendous ligand diversity
within the library, there is theoretically a ligand for every protein,
antibody, and peptide present in the starting material (
8, 9
). The
equalizer beads, as their name implies, work by binding proteins
until saturation is reached. The high-abundant proteins will reach
saturation quickly, while the lower-abundant proteins continue to
bind. The excess unbound proteins will be washed away in the
procedure. This results in a dramatic depletion of the most abun-
dant proteins, with a concurrent concentration of the middle- to
low-abundant proteins (
8, 9
).
In this chapter, we describe the preparation of ProteoMiner™-
treated human serum for 2D DIGE analysis.
2. Materials
Prepare all solutions using ultrapure water and use analytical grade
chemicals/reagents.
2.1. Equipment
1.
Class II biohazard fl ow hood.
2.
Rotary shaker.
3.
Refrigerated centrifuge (with a centrifugation force of at least
12,000 ×
g
).
4.
Spectrophotometer.
5.
IPGphor isoelectric focusing (IEF) unit (GE Healthcare,
Princeton, NJ, USA).
6.
Mini-PROTEAN 3 electrophoresis system (Bio-Rad, Hercules,
CA, USA).
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