Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 15)

Fig. 2.8. Albumin and IgG removal from serum using the Aurum
serum protein mini kit. Serum proteins were separated by 2-D
electrophoresis before and after treatment with an Aurum serum
protein mini column. Albumin and IgG are removed following treatment
with the column, improving resolution of other protein species.
Samples (100 µg) were focused on 11 cm ReadyStrip pH 5–8 IPG
strips, then run on 8–16% gels.
Before
Albumin
Heavy-chain IgG
Light-chain IgG
After
Albumin
Fig. 2.7. Clean fractionation by pI. Mouse liver extract was
fractionated using the MicroRotofor cell. 2-D separations of the
unfractionated sample (120 µg) and fractions (30 µg) are shown.
Prior to 2-D separation, samples were treated with the ReadyPrep
2-D cleanup kit to remove extra ampholytes. Note the clean pH
boundaries of fraction 3 and the enrichment of proteins in the pH
region it covers.
Fraction 3, pH 6.04
Unfractionated
Fraction 3, pH 6.04
pH 4.7 5.9
pH 3 10
pH 3 10
Products for Fractionation by pI
Products for Fractionation by Size (MW)
Products for Depletion
The Rotofor ® , Mini Rotofor, and MicroRotofor cells
separate and concentrate proteins into discrete
fractions by liquid-phase IEF. Following ampholyte
removal and sample concentration with the
ReadyPrep 2-D cleanup kit, each of the resulting
liquid fractions can then be separated on narrow-
or micro-range IPG strips.
The Model 491 prep cell and mini prep cell
perform size-dependent high-resolution
fractionation of proteins by continuous-elution gel
electrophoresis (using native PAGE or SDS-PAGE).
The large sample capacity (50 µl–15 ml, and
0.5–500 mg protein) of these cells makes them
particularly effective tools for the enrichment of
low-abundance proteins (Zerefos et al. 2006,
Xixi et al. 2006, Fountoulakis et al. 2004).
Bio-Rad’s Aurum Affi-Gel ® Blue and Aurum serum
protein mini kits represent a simple, low-cost
alternative to immunodepletion. These kits use
affinity chromatography to easily and effectively
remove albumin (Affi-Gel Blue) or albumin and IgG
(serum protein kit) in a single spin column.
Model 491 Prep Cell and Mini Prep Cell
Rotofor Family of Liquid-Phase IEF Cells
Aurum Ion Exchange Kit
26 27
2-D Electrophoresis Guide Theory and Product Selection
Fractionation by Size (MW)
Size-dependent separation is a powerful fractionation
strategy in studies focused on a particular protein or
protein family and their posttranslational modifications
because these proteins tend to be of similar size
(Fountoulakis and Juranville 2003). Proteins can
be separated into size-dependent fractions by
polyacrylamide gel electrophoresis (PAGE), particularly
continuous-elution electrophoresis.
Chapter 2: Sample Preparation
Depletion and Dynamic Range Reduction
One of the major difficulties facing proteomics is the
issue of dynamic range, or the variation in abundance
among sample proteins that typically spans several
orders of magnitude. This range typically exceeds that
over which proteins can be effectively detected and
quantified. Various strategies have been developed
for the reduction of sample dynamic range, and
they have proven beneficial for the study of low-
abundance proteins.
Depletion
Samples may be dominated by a few abundant
proteins whose presence can obscure less abundant
proteins and limit the capacity and resolution of the
separation technique employed. This is particularly
apparent for serum and plasma; the study of lower-
abundance proteins from serum or plasma is
often complicated by the presence of albumin and
immunoglobulin G (IgG), which together contribute
up to 90% of the total protein in a serum sample.
These proteins obscure comigrating proteins and limit
the amount of total serum protein that can be loaded
on 2-D gels. To obtain meaningful results from serum
samples, these proteins must be removed (Figure 2.8).
A strategy for specific depletion of abundant proteins
by immunoaffinity chromatography has been widely
used (Pieper et al. 2003, Roche et al. 2009, Tu et al.
2010, Ichibangase et al. 2008). Though this method is
effective, the need for antibodies renders it expensive
and limits its applicability to the specific sample type
for which the antibodies were developed.