Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 13)

Products for Fractionation by
Subcellular Location
Each of the following kits produces a fraction
with a distinct protein composition:
■ ■
ReadyPrep protein extraction kit (signal) takes
advantage of the limited solubility of plasma
membrane microdomain structures (for example,
lipid rafts and caveolae) in nonionic detergents
at 4°C to yield a protein pellet that is enriched
in membrane-associated signalling proteins,
including glycophosphatidylinositol (GPI)-anchored
proteins, caveolin and associated proteins,
acetylated tyrosine kinases, and G proteins
(Simons and Ikonen 1997)
■ ■
ReadyPrep protein extraction kits (membrane I and
membrane II) use different techniques to isolate
integral membrane and membrane-associated
proteins without the need for density gradients.
The membrane I kit is based on temperature-
dependent partitioning of hydrophobic proteins
into the detergent-rich phase of a Triton X-114/
water two-phase system (Bordier 1981, Prime
et al. 2000, Santoni et al. 2000). It is a quick
and effective protocol for enriching membrane
proteins without the need for ultracentrifugation.
More complex membrane proteins (those with
larger numbers of transmembrane domains) are
better isolated using the membrane II kit, which
enriches integral membrane proteins by treating
a membrane preparation with sodium carbonate
(Fujiki et al. 1982, Molloy et al. 2000); this protocol
requires ultracentrifugation
■ ■
ReadyPrep protein extraction kit (cytoplasmic/
nuclear) uses a proprietary buffer and differential
centrifugation to isolate intact nuclei and a strongly
chaotropic extraction buffer to quickly prepare
highly enriched fractions of cytoplasmic and
nuclear proteins from eukaryotic samples
Fig. 2.4. Differences in 2-D patterns obtained using ReadyPrep protein extraction kits: signal (A), membrane I (B), and membrane II (C)
kits. Mouse liver samples were extracted using each kit, and purified proteins were separated using 17 cm ReadyStrip pH 3–10 NL IPG strips
and 8–16% gels. Overall spot patterns differ for A, B, and C even though all three kits isolate membrane proteins, indicating that each kit isolates
different sets of proteins.
A B C
ReadyPrep Protein Extraction Kit
22 23
2-D Electrophoresis Guide Theory and Product Selection Chapter 2: Sample Preparation
Prefractionation
Proteomic analysis is often applied to samples that
have undergone prior fractionation (prefractionation),
and the reasons for this are varied. In cases where
only a defined subset of the proteome is under
study, prefractionation can increase the chances
of meaningful discovery by removing proteins not
likely to be of interest from the sample. For example,
in studies of mitochondrial processes, it is sensible
to perform the proteomic analysis on a subcellular
fraction enriched in mitochondria. In other cases,
specific proteins of interest may be enriched through
fractionation and analyzed by 2-D electrophoresis
in the absence of potentially interfering proteins.
Prefractionation can also be used to separate a
sample into multiple fractions of lower complexity
that can then be analyzed separately; this can
enable identification of lower-abundance proteins
that might otherwise be undetectable in the
unfractionated sample.
Prefractionation increases the depth of proteome
analysis, but it does so at the expense of a greater
workload and reduced throughput. Try to use a
fractionation method that generates minimal protein
overlap between fractions.
Proteins can be fractionated by a number of different
techniques. The choice of method depends on
the sample, experimental goals, and available
instrumentation:
■ ■
Chemical and centrifugal methods —
use of selective precipitation or selective extraction
or centrifugation steps to separate proteins or
partition different subcellular compartments.
In many instances, protein extraction protocols
can incorporate fractionation steps through the
selective use of certain chemical reagents
■ ■
Electrophoretic methods — application of
liquid-phase IEF or preparative SDS-PAGE with
the goal of protein enrichment. Though neither
of these techniques is orthogonal to either of the
two dimensions employed in 2-D electrophoresis
and neither offers additional resolving power to
the analysis, electrophoresis has proven useful in
allowing the enrichment of low-abundance proteins.
A protein in a size- or pI-enriched fraction can be
subjected to 2-D electrophoresis at a higher amount
relative to the unfractionated sample, allowing the
analysis of proteins present below detection levels
(Zuo and Speicher 2000, Fountoulakis and
Juranville 2003)
■ ■
Chromatographic methods — use of
chromatographic separation principles to enrich
low-abundance proteins or generate fractions of
reduced complexity (Fountoulakis et al. 1997,
Badock et al. 2001, Butt et al. 2001, Smith et al.
2004, Qin et al. 2005, Yuan and Desiderio 2005).
Virtually any chromatographic procedure can be
used as a prefractionation step; examples include
size exclusion, affinity, ion exchange, and reverse-
phase resins
Using these methods alone or in combination, proteins
can be separated upstream of 2-D electrophoresis
(prefractionated) by their physical or chemical
properties, as described below. Some of these
methods, however, may introduce ionic or other
contaminants that must be removed before IEF.
Also, increasing the number of sample handling steps
may increase variability and the risk of sample loss.
Fractionation by Subcellular Location
There are many techniques for preparing fractions
enriched in subcellular organelles or membrane
types, and there are several examples in which these
techniques have been used to prepare samples for
2-D electrophoresis and other proteomic analyses
(Huber et al. 2003). Methods for organellar
fractionation generally involve differential and density
gradient centrifugation (Stasyk et al. 2007, Fialka et
al. 1997). However, fractionation schemes involving
aqueous polymer phase separation (Tang et al.
2008) and free-flow electrophoresis (Zischka et al.
2003, Eubel et al. 2008) have been described for this
purpose as well. These methods are usually specific
for the source material (cells or tissue). In some cases,
fractions representing different subcellular sites can
be generated on the basis of solubility under different
conditions (see the Fractionation by Solubility/
Hydrophobicity section). These methods are
more general in application.
Bio-Rad offers several ReadyPrep protein extraction
kits for the isolation of fractions enriched in integral
membrane and transmembrane proteins (Figure 2.4),
as well as nuclear and cytoplasmic proteins (see the
Products for Fractionation by Subcellular Location
sidebar).