Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 9)

Products for Cell Lysis and
Protein Extraction
ReadyPrep ™ mini grinders contain a grinding
tube, grinding resin, and fitted pestle and offer an
easy, efficient mechanism for manually grinding
small biological samples. The grinding resin is a
neutral abrasive material made of a high-tensile
microparticle that does not bind protein or nucleic
acids. The combination of the pestle and resin
effectively disrupts animal or plant tissues and cells.
ReadyPrep mini grinders are available for purchase
separately or as components of the MicroRotofor
™
cell lysis kits.
Bio-Rad also offers a range of kits for cell disruption
and protein extraction:
■ ■
MicroRotofor lysis kits 1 provide convenient,
effective methods optimized for the preparation
of protein samples from mammalian, plant, yeast,
and bacterial sources. Depending on the sample
type, these kits employ tissue maceration using
ReadyPrep mini grinders and/or solubilization into
a chaotropic extraction buffer
■ ■
The ReadyPrep protein extraction kit (total protein)
uses the powerful zwitterionic detergent ASB-14
in a strongly chaotropic solubilization buffer to
prepare total cellular protein extracts suitable for
2-D electrophoresis (depending on sample type,
additional cell lysis protocols may be needed when
using this kit)
■ ■
Other ReadyPrep protein extraction kits facilitate
extraction of specific classes of proteins and are
discussed later in this chapter
Such standardized lysis and extraction protocols
are often useful for initial proteomic analyses and
for consistent sample preparation.
1 For added convenience, the extraction buffer included with these kits can also be used as the sample solution for IEF with the
MicroRotofor cell or with IPG strips.
Products for cell lysis and protein extraction. A number of other
ReadyPrep protein extraction kits facilitate disruption and extraction
of specific classes of proteins.
MicroRotofor Lysis Kit
ReadyPrep Mini Grinder
ReadyPrep Protein Extraction Kit
14 15
2-D Electrophoresis Guide Theory and Product Selection Chapter 2: Sample Preparation
Protein Solubilization
Proteins in a biological sample are often associated
with other proteins, integrated into membranes, or
parts of large complexes. Protein solubilization is the
process of breaking interactions involved in protein
aggregation (Rabilloud 1996), which include disulfide
and hydrogen bonds, van der Waals forces, and ionic
and hydrophobic interactions. If these interactions are
not disrupted, proteins can aggregate or precipitate,
resulting in artifacts or sample loss. For successful
2-D electrophoresis, proteins must be well solubilized.
Sample lysis solutions typically contain a number
of compounds that meet the requirements, both
electrically and chemically, for compatibility with IEF.
To allow high voltages to be applied during IEF without
producing high currents, the compounds must not
increase the ionic strength of the solution. In some
cases, it may be necessary to prepare samples using
additives that facilitate protein solubilization but that
have limited compatibility with IEF (for example, salts
and SDS). In these cases, the potentially interfering
substance must be removed prior to sample
application, or actions must be taken to mitigate its
effect (see the Removal of Interfering Substances
section). See Chapter 9 for sample preparation
procedures and solutions; for a thorough discussion
of solubilization methods, refer to Rabilloud (2000).
Chaotropic Agents
These compounds disrupt hydrogen bonds and
hydrophobic interactions both between and within
proteins. When used at high concentrations,
chaotropic agents disrupt secondary protein
structure and bring into solution proteins that are
otherwise insoluble. The neutral chaotropic agent
urea is used at 5–9 M, often with up to 2 M thiourea,
which can dramatically increase the number of
proteins solubilized (Rabilloud et al. 1997). Thiourea
is weakly soluble in water but more soluble in high
concentrations of urea; therefore, a mixture of 2 M
thiourea and 5–8 M urea is used when strongly
chaotropic conditions are required. Charged
chaotropic agents such as guanidine hydrochloride
are incompatible with IEF.
If using thiourea during sample preparation, also add it
to the first-dimension rehydration solution; otherwise,
the proteins that require thiourea for solubility will
come out of solution during IEF.
Urea and thiourea can hydrolyze to cyanate and
thiocyanate, respectively; these products modify
amino groups on proteins (carbamylation) and give
rise to artifactual charge heterogeneity. Since heat
promotes this hydrolytic reaction, never heat urea-
or thiourea-containing solutions above 37°C in the
presence of protein (McCarthy et al. 2003).
Detergents
Detergents disrupt hydrophobic interactions between
and within proteins and are classified as neutral,
zwitterionic, anionic, and cationic (Luche et al. 2003).
Some proteins, especially membrane proteins, require
detergents for solubilization during isolation and for
maintaining solubility during IEF.
Sample preparation for 2-D electrophoresis commonly
uses neutral or zwitterionic (having both positive and
negative charges resulting in a neutral net charge)
detergents at concentrations of 1–4%, since these
detergents do not introduce a net charge and therefore
allow proteins to migrate at their own charges during
IEF. Examples of neutral detergents include Tween,
octylglucoside, dodecyl maltoside, Triton X-100, and
Triton X-114. Examples of zwitterionic detergents
include CHAPS, CHAPSO, ASB-14, and SB 3-10.
In practice, only a few detergents are used in IEF
(Table 2.2). With few exceptions, only a single
detergent should be used because the effects of
detergents are not additive and can be unpredictable
in combination. Anionic and cationic detergents
are generally not suitable for IEF.
SDS is unparalleled in its ability to efficiently
and rapidly solubilize proteins. Although SDS is
incompatible with IEF as an anionic detergent, it can
be used in the initial preparation of concentrated
protein samples. In these cases, another IEF-
compatible detergent must be used in excess to
disrupt the binding of SDS to protein (Ames and
Nikaido 1976). Also to be considered is how the
detergent interacts with high concentrations of
urea. When using SB 3-10, for example, the urea
concentration is limited to 5 M, but ASB-14 can be
used with 9 M urea (Chevallet et al. 1998).