Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 16)

Fig. 2.9. The ProteoMiner protein enrichment kit improves
resolution and spot counts in 2-D gels. In an untreated sample,
albumin and other high-abundance proteins dominate the gel and
obscure signals from less abundant proteins. On a gel generated
using an equal amount of total protein from a treated serum sample,
however, resolution is dramatically improved and a greater number
of protein spots is visualized.
Untreated
Treated
ProteoMiner Technology
ProteoMiner technology employs a combinatorial
library of hexapeptides bound to a chromatographic
support. Combinatorial synthesis creates a large library
of unique hexapeptides, with each hexapeptide bound
to a stationary support, or bead. Each bead, featuring
a unique ligand, is expected to bind specifically to one
or a small number of different proteins in a mixture,
and the library of all possible sequences binds proteins
up to the capacity of available beads. When a complex biological sample is applied to the
beads, high-abundance proteins saturate their ligands
and excess proteins are washed away. In contrast,
low-abundance proteins do not saturate their binding
sites. Therefore, different samples retain relative
expression levels similar to the original samples.
Moreover, low-abundance proteins are enriched if the
beads are eluted in a volume smaller than the original
sample. The overall effect of ProteoMiner technology
results in the bound and eluted material consisting of a
significantly lower amount of total protein, thus allowing
resolution of a greater diversity of species.
Biological sample
(large dynamic range)
Ligand library
Bind mixture
to library
Wash away
unbound protein
Elute bound
sample for analysis
Depletion of plasma and serum samples by ProteoMiner technology. Each bead features a different hexapeptide ligand with affinity
for specific proteins in a sample. Samples are applied to the beads, allowing proteins to bind to their specific ligands. Proteins in excess are
washed away, and those proteins bound to the beads are eventually eluted, allowing further downstream analysis.
28 29
2-D Electrophoresis Guide Theory and Product Selection Chapter 2: Sample Preparation
Dynamic Range Reduction
ProteoMiner ™ protein enrichment technology uses a
bead-based library of combinatorial peptide ligands
that act as unique binders for proteins (Thalusiraman
et al. 2005, Guerrier et al. 2006). When a complex
sample is applied to the beads, abundant proteins
saturate their specific ligands while the remaining
proteins can be washed away. Low-abundance
proteins are concentrated on their specific ligands
and will be enriched relative to the abundant proteins
following elution. In contrast to immunodepletion,
ProteoMiner has no intrinsic specificity for any
particular sample type and can be used to decrease
high-abundance proteins in any sample that could
benefit from such a treatment. The technology has
been most widely applied to serum and plasma
(Sennels et al. 2007); however, several examples
of successful application of ProteoMiner to other
samples have also been reported (Castagna et al.
2005, Guerrier et al. 2007, D’Ambrosio et al. 2008,
Bandhakavi et al. 2009).
ProteoMiner protein enrichment kits:
■ ■
Decrease the amount of high-abundance proteins
without immunodepletion, preventing the loss of
proteins bound to high-abundance proteins
■ ■
Enrich medium- and low-abundance proteins that
cannot be detected through traditional methods
(Figure 2.9)
■ ■
Do not rely on a predefined set of antibodies,
unlike immunodepletion products
■ ■
Are compatible with a variety of sample types
■ ■
Offer a convenient, easy-to-use format
■ ■
Can be used for differential expression analysis