1. health and fitness
2. disease
3. aids and hiv

1408
DOI 10.1002/pmic.200300471
Proteomics 2003, 3, 1408–1417
Review
Margaret M. Shaw1, 2
Beat M. Riederer1, 2
Sample preparation for two-dimensional gel
electrophoresis
1
Institut de Biologie Cellulaire
et de Morphologie,
Université de Lausanne,
Lausanne, Switzerland
2
Centre de Neurosciences
Psychiatriques,
Hôpital de CERY,
Prilly, Switzerland
The choice of sample preparation protocol is a critical influential factor for isoelectric
focusing which in turn affects the two-dimensional gel result in terms of quality and
protein species distribution. The optimal protocol varies depending on the nature of
the sample for analysis and the properties of the constituent protein species (hydrophobicity, tendency to form aggregates, copy number) intended for resolution. This
review explains the standard sample buffer constituents and illustrates a series of protocols for processing diverse samples for two-dimensional gel electrophoresis, including hydrophobic membrane proteins. Current methods for concentrating lower abundance proteins, by removal of high abundance proteins, are also outlined. Finally, since
protein staining is becoming increasingly incorporated into the sample preparation
procedure, we describe the principles and applications of current (and future) pre-electrophoretic labelling methods.
Keywords: Detergents / Prefractionation / Prelabelling / Protease inhibitors / Review / Thiourea /
Two-dimensional gel electrophoresis
PRO 0471
Contents
1
2
2.1
2.2
2.3
2.4
2.5
2.6
3
3.1
3.2
3.3
4
5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample buffer constitutents . . . . . . . . . . . . .
Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thiourea . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Detergents . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reducing agents . . . . . . . . . . . . . . . . . . . . . .
Carrier amphloytes/Immobilines . . . . . . . . . .
Protease inhibitors . . . . . . . . . . . . . . . . . . . . .
Recent advances in sample preparation
methodology . . . . . . . . . . . . . . . . . . . . . . . . .
Sample prefractionation . . . . . . . . . . . . . . . .
Resolution of “elusive” proteins . . . . . . . . . .
Prelabelling applications . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 Introduction
1408
1409
1409
1409
1410
1410
1411
1411
1412
1412
1414
1415
1416
1416
Correspondence: Dr. B. M. Riederer, IBCM, Rue du Bugnon 9,
1005 Lausanne, Switzerland
E-mail: [email protected]
Fax: 141-21-692-5105
Abbreviations: ASB14, myristic amidosulphobetaine; SB3-10,
caprylyl sulphobetaine; TBP, tributyl phosphine; TCEP, Tris (2carboxyethyl) phosphine
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Two-dimensional gel electrophoresis (2-DE) is a protein
separation technique that combines two different electrophoretic methods, that is gel IEF in the first dimension (in
which proteins are separated according to pI) and SDSPAGE in the second dimension (separation according to
molecular weight (Mr)).
The aim of sample preparation for 2-DE is to convert the
native sample into a suitable physicochemical state for
first dimension IEF while preserving the native charge
and Mr of the constituent proteins. In many cases this
means that the proteins of the sample need to be solubilised, disaggregated, denatured and reduced. However,
the specific sample preparation method depends first
and foremost on the aim of the separation. For example,
if the aim of the first dimension is to resolve the isoelectric
points of soluble aggregates, then the IEF needs to be
carried out under nondenaturing, nonreducing conditions
and the aim of the sample preparation method would be
to achieve protein solubilisation without disaggregation.
In many cases solubilisation under these conditions is
incomplete and only the supernatant from the centrifuged
sample can be applied to the first dimension. In nondenaturing, nonreducing IEF partial solubilisation of thylakoid
membrane proteins has been achieved with the nonionic
Proteomics 2003, 3, 1408–1417
detergents Triton X-100 and N-dodecyl-b-D-maltoside [1].
Triton X-100 and dodecyl maltoside are considered nondenaturing as they are more efficient at breaking lipid-lipid
and lipid-protein interactions than protein-protein interactions.
Sample preparation
1409
Probably the aim of most 2-D gel applications is, however,
to resolve as many proteins as possible within a particular
pI/Mr range, to facilitate comparison of two sets of data
samples. In this case, it is preferable to carry out IEF
under denaturing and reducing conditions. The following
gives an account of the individual sample buffer constituents, their function, possible problems associated with
each buffer component and recent advances or modifications which improve the final 2-D gel outcome and facilitate representation of a certain species of proteins which,
due to their physicochemical properties, have until
recently, remained unresolved on the 2-D gel map.
10 M urea will degrade to equilibrium to form 20 mM
cyanate. Reaction of cyanate ions with the amine
groups on proteins (carbamylation) removes the positive
charge on the amine, which affects the IEF result. Carbamylation is not limited to amine groups. Lippincott
and Apostol [2] investigated carbamylation of sulphhydryl groups on cysteine residues and found that carbamylation of cysteines readily occurs under slightly acidic
conditions (pH 6) but that carbamylmercaptans are
unstable in an alkaline environment. The issue of carbamylation presents a potential problem when IPG strips
are rehydrated overnight with the prepared sample in
strip rehydration solution. However, the overall quality
of the 2-D gel map only appears to be affected at
extended strip rehydration times of around 24 h [3]. To
reduce the effects of carbamylation the cyanate scavenger, spermine, can be added to the sample solution or
strip reswelling solution [4].
2 Sample buffer constitutents
2.2 Thiourea
2.1 Urea
Use of thiourea in combination with urea for IEF was first
reported by Thierry Rabilloud in 1996 at the Siena 2-D
electrophoresis conference [5]. As a result a whole variety of proteins previously elusive to the 2-D gel map
could be resolved [6–8]. Thiourea has particularly been
shown to improve solubilisation of hydrophobic membrane proteins [9, 10]. One disadvantage of thiourea,
reported by Galvani et al. [11], is that at pH values of
8.5–9, which coincide with the pH of the buffer for equilibration after the first dimension (pH 8.8), the sulphur
atom of thiourea is as reactive as the SH group of cysteine. As a consequence thiourea present in the first dimension gel scavenges the iodoacetamide during equilibration after the first dimension resulting in poor alkylation of proteins. To counteract this, Galvani et al. [11]
recommend that thiourea should first be omitted from
the solubilising solution and incorporated after the sample has been reduced and alkylated, prior to first dimension IEF. Reduction and alkylation at the sample preparation stage has the additional advantage of inhibiting
carbamylation since (i) DTT acts as an effective scavenger of cyanate, and (ii) alkylation prevents further reaction with cyanate ions [2]. Thiourea is generally used at
concentrations of 2 M in conjunction with 5–7 M urea although Musante et al. [7] reported a reduction in protein
resolution associated with increasing concentrations of
thiourea and consequently limited the concentration of
thiourea to 0.5 M. The loss in protein resolution may possibly be a result of poor transfer to the second dimension, as thiourea additionally tends to inhibit SDS-protein
binding, or of improved solubilisation of lipids, which
affect resolution on 2-D gels.
Sample solutions for first dimension separations under
denaturing conditions always include urea. This neutral
chaotrope denatures proteins by disrupting noncovalent
and ionic bonds between amino acid residues. Its neutral
charge renders it ideal for IEF as it remains in the gel during focusing and does not migrate. It may be included in
the sample solution at a concentration of ,5 M to as high
as 9.8 M. However, urea solutions are not stable. Spontaneous degradation of urea to cyanate (Fig. 1) occurs at
room temperature. If left overnight at room temperature
Figure 1. Cyanate formation and carbamylation.
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1410
M. M. Shaw and B. M. Riederer
2.3 Detergents
Traditionally, sample buffer protocols for 2-DE included
the anionic detergent SDS [12]. Since protein-detergent
micelles formed on SDS treatment carry an overall negative charge, inclusion of SDS in sample buffers for IEF
seems illogical. However, SDS was found not to interfere
in IEF if used together with an excess of nonionic or zwitterionic detergent (e.g. NP-40, Triton X-100, CHAPS) and
it was suggested by O’Farrell [12] (who used NP-40 in
conjunction with SDS) that the nonionic detergent forms
mixed micelles with the SDS which migrate to the anode.
Ames and Nikaido [13] investigated the criteria for SDS
compatibility and established that the NP-40:SDS concentration ratio should be at least 8:1 in order to avoid a
streaking effect. In addition the final concentration of SDS
in the IEF sample buffer should be below 0.25% [14].
Some inconveniences of using SDS for protein solubilisation for 2-DE application are discussed in a review by Molloy [5] who also mentions that the electrophoretic removal
of SDS from the proteins during focusing may in some
cases result in in-gel isoelectric precipitation. Indeed,
even small amounts of SDS have been found to remove
all the detergent from the gel due to the formation and anodic migration of mixed micelles leaving the proteins
exposed to an environment containing little or no detergent [15]. An additional factor concerning SDS solubilisation/denaturation is that SDS is not suitable for all applications, since strongly acidic proteins do not bind SDS.
The alternative for such proteins would be to use cationic
detergents, which are, however, not widely investigated in
2-DE applications.
Proteomics 2003, 3, 1408–1417
form inclusion compounds with urea, for example, SB3–
10 is a powerful surfactant which is however, compatible
only with low concentrations of urea, whereas the shortertailed, less efficient detergent, SB3–8, is compatible with
high concentrations of urea [19]. ASB14 is, however, an
efficient surfactant compatible with 9 M urea. Structures
of several nonionic and zwitterionic detergents are shown
in Fig. 2.
2.4 Reducing agents
Reduction of disulphide bonds aids solubilisation of complex mixtures of proteins. This is commonly achieved with
free thiol-containing reducing agents such as dithiothreitol (DTT), dithioerythritol (DTE) or 2-mercaptoethanol [20].
However, 2-mercaptoethanol is no longer widely used in
carrier ampholyte IEF, particularly where basic proteins
are of interest, since it ionises at the alkaline end of the
gel and is driven electrophoretically along the pH gradient, apparently sweeping away focused carrier ampholytes, resulting in a disturbance in this region [21]. In addition, DTT, DTE and an alternative reducing agent, tris(2carboxyethyl) phosphine (TCEP), which are also charged
at alkaline pH, migrate towards the anode, depleting the
basic end of the gel during IEF, which results in re-oxidation of reduced S-S bonds [22]. In the absence of an alkylation step during sample preparation this results in precipitation of some proteins (particularly keratins and keratin-associated proteins from hair and wool which are rich
in disulphide bonds) and often in spurious spots in the
alkaline pH region due to formation of “scrambled” disul-
Nonionic detergents are favoured for nondenaturing IEF
as most, such as Tween 80, NP-40 and Triton X-100 are
mild detergents which retain enzyme activities. The original protocols of O’Farrell [12] and Klose [16] utilised NP40 and Triton X-100. These detergents are generally used
at concentrations between 0.4–4%, although as high as
10% NP-40 has been used in sample buffers by Lenstra
and Bloemendal [17]. N-dodecyl-b-D-maltoside is also a
nonionic detergent which has been used at a final concentration of 0.47% [1].
The zwitterionic detergent, CHAPS has since been
demonstrated to be more effective at solubilisation [18].
CHAPS is currently the most commonly used detergent
in standard procedure for 2-DE and belongs to the class
of linear sulphobetaine surfactants which also include
caprylyl sulphobetaine (SB3–10), and amidosulphobetaine 14 (ASB14).
Urea tolerance is a factor which must be taken into
account when selecting detergents for IEF sample buffers. Some of the powerful detergents disadvantageously
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Nonionic and zwitterionic detergents.
Proteomics 2003, 3, 1408–1417
Sample preparation
phide bridges [23]. The noncharged reducing agent tributyl phosphine (TBP) overcomes this problem. DTT and
DTE are both used in the concentration range of 20–
100 mM, whereas higher concentrations are required for
the less potent 2-mercaptoethanol. TBP has been used
at a concentration of 2 mM [24].
2.5 Carrier ampholytes/Immobilines
The addition of carrier ampholytes to the solubilising buffer has several advantages. First, where IPG strips are
used, carrier ampholytes are useful in inhibiting interactions between hydrophobic proteins and Immobilines
which tend to occur at the basic end of the gel, leading
to a streaking effect due to precipitation [25, 26]. Carrier
ampholytes additionally scavenge cyanate ions and help
in the precipitation of nucleic acids during centrifugation.
Carrier ampholytes are generally used at a concentration
of 0.5–2%, although 2% is normally only recommended if
protein solubilisation is a problem. Table 1 provides a list
of different sample buffers used for solubilising various
specimens for 2-DE.
1411
2.6 Protease inhibitors
The denaturing properties of most sample buffers are
often sufficient to inhibit the action of proteases [3, 20].
However, cathepsin C, several carboxypeptidases and
endoproteinases, chymotrypsin, trypsin, plasmin and
proteinase K are examples of some proteases which
remain active in 1 mg/mL SDS. Thus, depending on the
sample buffer employed, inclusion of protease inhibitors
may be necessary. In addition, protease inhibitors are
useful if lengthy sample manipulations are carried out
prior to the first dimension loading. This is exemplified by
the work of Olivieri et al. [33] who observed that failure to
add protease inhibitors during the lengthy procedure of
erythrocyte membrane preparation, which is carried out
in physiological buffers providing favourable conditions
for protease activity, led to massive degradation of high
molecular weight proteins, resulting in a mixture of low
molecular mass (,50 kDa) proteins and peptides on the
2-D gel map. Some commercially available protease inhibitors and their target proteases are summarised in
Table 2.
Table 1. Examples of solubilisation buffers which have been used for preparation of different samples for 2-DE.
Sample
Urea
Thiourea
Reducing
agent
Chinese Hamster
Ovary cells
(exogenously
expressing membrane
proteins)
7M
2M
Mouse brain
5M
Carrier
ampholytes
Reference
50 mM
2% C80
DTT
or
2 mM TCEP- 2% CHAPS
or
HCl
2% ASB14
0.5% (3–10)
Henningsen et
al. [27]
2M
60 mM
DTT
2% CHAPS
2% (3–10)
Riederer and
Shaw [3]
7M
Bacterial outer
membrane proteins (after
carbonate
washing)
7M
E. coli outer
membrane proteins
(after carbonate
washing)
2M
30 mM
DTT
1% ASB14 0.5%
Triton
X-100
0.5% (3–10)
Molloy et al. [28]
2M
2 mM
TBP
1% ASB14
0.5% (3–10)
Molloy et al. [29]
Human plasma
8M
None
10 mM
DTE
2% CHAPS
0.8% (4–8)
Liberatori et al.
[30]
Mouse liver
8M
None
60 mM
DTT
0.5% Triton
X-100
2% (3–10)
O’Connell and
Stults [31]
Disaggregated
human kidney
tissue
9M
None
65 mM
DTE
4% CHAPS
4% (9–11)
Sarto et al. [32]
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Detergent
1412
M. M. Shaw and B. M. Riederer
Proteomics 2003, 3, 1408–1417
Table 2. Protease inhibitors and their targets
Protease inhibitor
Target
Recommended
working
concentration
APMSF
Plasma serine
proteases
10–20 mM
Aprotinin
Serine proteases
0.01–0.3 mM
Bestatin
Aminopeptidases
40 mg/mL
Dichloroisocoumarin
Serine proteases
1–43 mg/mL
Disodium EDTA
Metalloproteases
100 mM
E-64
Thiol proteases
1.4–2.8 mM
Leupeptin
Serine and thiol
proteases
1 mM
Pepstatin
Acidic proteases
1 mM
PMSF
Serine proteases
100–1000 mM
Phosphoramidon
Thermolysin
Collagenase
Metalloendoproteases
7–569 mM
TLCK.HCl
Trypsin
Thiol proteases
37–50 mg/mL
TPCK
Chymotrypsin
Thiol proteases
70–100 mg/mL
3 Recent advances in sample preparation
methodology
There are three areas in sample preparation methodology
which have been the subject of significant advance in
recent years. These are (1) sample prefractionation, (2)
resolution of “elusive” proteins, and (3) prelabelling applications.
3.1 Sample prefractionation
Several examples can be provided of comparative 2-D
gel studies in vivo where little or no differences have
been observed in the 2-D gel maps between the control
and test groups (Riederer and Shaw, unpublished data;
Hondermarck et al. [34]). In many cases, even after some
anatomical prefractionation (for example, removal of hippocampus or prefrontal cortex from whole brain) such
studies fail to show differences due to the heterogeneity
of cell types within a specimen: a protein marginally upregulated in one cell type by a particular drug treatment
may be missed due to the majority of other unaffected
cell types in the same sample. Similarly, some proteins in
the sample may comigrate to the same spot, clouding the
result. This has often led researchers to abandon the in
vivo study in favour of a homogenous cell line. However,
examples of enrichment of cells according to specific
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
type do exist: fluorescence-activated cell sorting (FACS)
of antibody-bound cells enables the collection of specific
cell types from clinical specimens. One unique example
which does not require antibody binding is the isolation
of rat b-cells which can be distinguished from other cell
types by their autofluorescence. After trypsin digestion
of islets, rat b-cells can be FACS-sorted to .95% purity.
Some cell types, for example, fibroblasts, neurons or glial
cells can be “enriched” from clinical specimens by producing primary cultures under conditions which select
out the cell type of interest and inhibit growth of other
cell types. However, these manipulations are likely to
result in activation of stress and other signalling pathways
which may induce a considerable change in the protein
profile of the cell.
Subcellular fractionation either from a homogeneous cell
type or from tissues composed of heterogeneous cell
types provides an additional method of protein exclusion.
Classical subcellular fractionation procedures were
based mainly on centrifugation techniques [35–37]. However, recently developed methods rely more and more on
differential resistance or susceptibility of cell components
to various extraction buffers, for example the resistance
of actin filaments, intermediate filaments and nuclei
against nonionic detergents, the resistance of most intermediate-sized filament proteins, vimentin and nuclear
proteins against the weakly ionic detergent sodium deoxycholate (in contrast to actin filaments which are susceptible to solubilisation by this detergent) and the resistance of intermediate-sized filaments against nonionic
detergents and 1.5 M KCl [17]. Similarly, Molloy et al. [9]
were able to partition Escherichia coli membrane proteins
from other proteins based on the limited solubility of
membrane proteins in solutions conventionally used for
IEF (8 M urea, 4% CHAPS, 100 mM DTT, 40 mM Tris base,
0.5% v/v Pharmalyte 3–10, 150 U endonuclease, pH 9.5)
and their partial solubility in a lysis buffer consisting of 5 M
urea, 2 M thiourea, 2 mM TBP, 2% w/v SB3–10, 2% w/v
CHAPS, 40 mM Tris base, 0.5% v/v Pharmalyte 3–10,
150 U endonuclease, pH 9.5. By a similar technique
André et al. [38] were able to purify intermediate filaments
from eukaryotic cells.
Using several independent chemical extraction methods,
Lenstra and Bloemendal [17] were able to characterise
seven distinct subcellular fractions from cultured hamster
lens cells on 2-D gels which represented the total detectable protein population of these cells (water soluble proteins, membrane proteins, actin filaments, intermediatesized filaments, microtubular proteins, polyribosomes
and nuclei). For 2-D gel analysis, these fractions were dissolved in SDS-containing lysis buffer [39] and, despite the
recommendations of Wilson et al. [40], boiled for 3 min,
Proteomics 2003, 3, 1408–1417
then brought to 9 M urea 10% NP-40, 5% v/v Ampholines
pH 3–10 and 5% 2-mercaptoethanol. The sum of the 2-D
gel profiles of these fractions almost completely complemented the 2-D gel map of total cell lysates prepared by
freeze-thawing in MgCl2 (5 mM), KCl (25 mM), 50 mM Tris
HCl pH 7.4, treated with DNase I for 15 min at room temperature and diluting with SDS lysis buffer [39].
In many cases abundant proteins present a problem on
2-D gel maps as they severely limit the amount of nonabundant protein which can be loaded onto the first dimension and in addition, the enlarged spots which they
produce on the gel may cloud or displace other spots,
resulting in inaccurate pI/Mr representations of particular
proteins or failure to detect proteins which may be significant.
An example of a highly abundant protein in serum is albumin, which may constitute 50% of proteins present in
serum. The product ProtoClear for albumin removal [41]
comprised an affinity column with a monoclonal antibody
to albumin, allowing better representation of proteins
other than albumin on 2-DE gels. However, this product
is no longer available. Albumin removal columns are, however, offered by several companies, but these do not exclude some nonspecific binding. Fibrinogen is also a highly
abundant protein in plasma, which can be removed by
affinity chromatography. PlasmaSelect (Martinsried, Germany) has developed a fibrinogen adsorption system
(“Rheosorb”) consisting of a fibrinogen-specific pentapeptide ligand which selectively removes fibrinogen and
fibrin from plasma [42]. The basis of this ligand is currently
used by Geneva Proteomics (GeneProt, Geneva, Switzerland) for fibrinogen removal prior to 2-DE.
In addition to columns for removal of high abundance proteins, there also exist commercially available kits for
removal of substances which might otherwise interfere
in IEF (e.g. PlusOne 2-DE clean up kit, Amersham Biosciences, Little Chalfont, Bucks, UK). Interfering substances include salts, nucleic acids, lipids and polysaccharides. In some instances salts may induce protein
modification [43]. Generally, though, salts delay the onset
of protein focusing which cannot occur until the ions have
migrated to the electrodes. In addition salts increase the
conductivity of the IEF gel, as the current increases since
resistance is a constant, focusing occurs at a lower voltage, prolonging the time required for IEF. High salt concentrations may also cause electroendosmosis (EEO)
resulting in uneven water distribution in the gel which
forms zones of dehydration and overhydration. The salt
concentration for IPG strips when sample is applied by
in-gel rehydration should be lower than 10 mM. Application via sample cups permits higher salt concentrations
(up to 50 mM). However, proteins may precipitate at the
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample preparation
1413
sample application site as they move into the lower
salt environment of the first dimension gel [43]. Salts can
be removed from samples by dialysis, gel filtration, TCA
precipitation or use of protein concentration devices (e.g.
Centricon columns, Millipore, Walford, UK). Urine and
sweat are examples of clinical samples with a high salt
content. Urine sample preparation and solubilisation for
2-DE have been described by Anderson et al. [44] and
Edwards et al. [45].
Lipids bind proteins via hydrophobic interactions, affecting their charge and Mr. In many cases the protein lipid
complex is insoluble in aqueous solution, resulting in failure to enter the first dimension gel. This interaction can be
outcompeted by the addition of excess detergent. In the
case of lipid-rich tissues such as brain or adipose tissue,
excess lipids can be removed by acetone precipitation.
Polysaccharides can interfere in IEF by obstructing gel
pores. Ultracentrifugation removes high Mr polysaccharides. Lower Mr polysaccharides may be removed by precipitation in TCA, ammonium sulphate or phenol/ammonium acetate.
Nucleic acid rich samples usually constitute rapidly dividing cells, cultured cells or biopsies from fast-growing
tumours. Nucleic acids can bind proteins through electrostatic interactions, preventing focusing. High Mr nucleic
acids can additionally clog the pores of the acrylamide matrix. They can be removed by treatment with
nucleases, addition of carrier ampholytes with subsequent ultracentrifugation [12] or by precipitation with a
basic polyamine at high pH [46].
In addition to the removal of abundant proteins such
as albumin, column chromatography is also useful for
the removal of certain contaminants. Desalting columns,
for example, function by size exclusion. In addition to
size exclusion chromatography, several other chromatographic techniques (hydrophobic interaction and reversed-phase chromatography, ion-exchange and affinity
chromatography) [47] can be applied to sample prefractionation for 2-DE. However, the chromatographic principle employed depends on the aim of the electrophoretic
separation.
If only one or a group of proteins are to be studied by
2-DE, enrichment may be carried out by chromatographic
methods. However, one technique which enables enrichment of many different proteins within a particular pI
range is preparative IEF. This is useful if proteins are to
be resolved afterwards on high resolution narrow range,
first dimension gels, due to the limit to the overall quantity
of protein which can be applied to the first dimension and
avoids wasteful application of proteins which are outside
of the pI range.
1414
M. M. Shaw and B. M. Riederer
Preparative IEF devices include the “Rotofor” [48], produced by Bio-Rad (Hercules, CA, USA), which is based
on a system proposed by Bier et al. [49], the “Isoprime”
from Amersham Biosciences (Uppsala, Sweden) based
on a development by Righetti et al. [50] and the “Gradiflow” from Gradipore, demonstrated by Corthals et al.
[51]. The Rotofor is a rotating chamber in which samples
are fractionated in solution by IEF. However, this apparatus has no separation barriers and fractions obtained are
not well-resolved, with cross-contamination of proteins
between fractionated pools. The Isoprime apparatus is a
multicompartment electrolyser where each compartment
is separated by a polyacrylamide gel membrane with a
specific pH produced by Immobilines incorporated into
the membranes. The Isoprime apparatus produces high
quality fractions. However, it was developed primarily for
large-scale separation of partially purified preparations,
not for fractionation of crude extracts. Zou and Speicher
[52] developed a solution IEF device based on a scaleddown volume version of the Isoprime apparatus for prefractionation of cell extracts, which also yielded wellresolved fractions. This has been further developed as
the microscale solution or “nusol” IEF technique [53]. A
further off-gel IEF unit, consisting of a fluid-filled multiwell
device placed on top of an IPG gel has been developed
by Michel et al. [54]. Proteins diffuse through the gel and
into the solution of each well. This effectively increases
the capacity of the IPG gel and provides protein at
fractionated pI ranges in solution for further manipulation. The system allows resolution to as high as 0.1 pH
unit.
3.2 Resolution of “elusive” proteins
There exist several classes of proteins which have
eluded resolution on 2-D gel maps and thus represent
some of the proteins which continue to remain beneath
the tip of the “proteome iceberg”. Proteins fail to be
resolved on 2-D gels for a number of reasons. They
either do not enter the first dimension gel, or they enter
the gel but precipitate and do not migrate, forming
streaks. Precipitation also makes transfer to the second
dimension difficult. Other proteins may be present at
such low levels that they escape detection or they may
be highly unstable and degrade so rapidly that they cannot be detected, factors which may be counteracted by
prefractionation or the use of protease inhibitors. Finally,
some proteins may not be resolvable on 2-D gels simply
because their pIs fall outside the range of the first dimension. The latter are exemplified by the very basic (pI .10)
ribosomal proteins and histones. This problem was
solved by developments which enabled extension of the
pI extremes of IPG strips [55]. The increased reverse
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2003, 3, 1408–1417
electroendosmotic flow in these strips necessitated the
inclusion of isopropanol and glycerol in the reswelling solution [4].
The failure of proteins to enter IEF gels may be due to several factors. One is size. The spectrins represent a group of
large (280 kDa) filamentous proteins which, when using
IPG strips in the first dimension, are lost from the 2-D map
unless active instead of passive sample hydration into the
IPG strip is carried out [33]. Another property, which affects
the ability of proteins to enter IEF gels or remain in solution
during focusing is solubility. Proteins which resist solubilisation in specific sample buffers are invariably hydrophobic membrane proteins, nuclear proteins and proteins
highly prone to aggregation such as tubulin and keratins.
Improved solubilisation of keratins can be achieved by
ensuring (and maintaining) maximal reduction of disulphide bonds. This may be achieved by alkylation immediately after reduction, or with the highly potent, noncharged reducing agent, TBP. Further improvements have
been made by the inclusion of thiourea; in the absence of
this strong chaotrope, tubulin and fibronectin are barely
visible on 2-D gels. The addition of zwitterionic amphiphilic
surfactants (e.g. CHAPS or SB3–10) has also improved
protein solublisation. In a study comparing the solubilisation properties of various sample buffers, Rabilloud et al.
[6] found that CHAPS-containing solutions consisting of
urea and thiourea were more effective at solubilising high
Mr integral membrane proteins than SB3–10-containing
solutions. However, since SB3–10 is not compatible with
high levels of urea, the urea concentration in this case had
to be limited to 5 M, whereas the CHAPS-containing solution contained 7 M urea. Therefore, a direct comparison of
the efficacy of the two surfactants was not made.
A similar indirect comparison was carried out by Molloy
et al. [56] who compared the solubilisation efficacies of
a buffer containing 2% CHAPS and 2% SB3–10 with a
buffer containing 1% ASB14. The remaining components
of both buffers were essentially the same, except that the
CHAPS/SB3–10 buffer contained 5 M urea and 2 M thiourea, whereas the ASB14 buffer contained 7 M urea and 2 M
thiourea. They found that the ASB14-containing buffer
produced larger spots on the 2-D gels, indicative of more
protein. However, this improved yield may also be a result
of the increased urea concentration used in conjunction
with this surfactant.
The alkyl aminosulphobetaine, ASB14, used in conjunction with urea and thiourea, resulted in the appearance
of several integral membrane proteins not previously
detected on 2-D maps [57]. Nouwens et al. [58] directly
compared two sample buffers (both containing 7 M urea,
2 M thiourea, 0.5% carrier ampholytes and 2 mM TBP),
one buffer additionally containing 2% CHAPS and 2%
Proteomics 2003, 3, 1408–1417
SB3–10 and the other additionally containing 1% ASB14.
The ASB14 buffer was superior at solubilising bacterial
membrane proteins.
A direct comparison has also been made between a solubilisation buffer containing 2 M thiourea, 7 M urea and
4% CHAPS and the same buffer additionally containing
the amphiphilic nondetergent sulphobetaine NDSB256
(Calbiochem, La Jolla, CA, USA) (at 5% concentration). It
was found that this dual inclusion of surfactants markedly
increased the number of spots present on the 2-D gel, in
particular high Mr spots [6].
Macri et al. [59] reported an increase in the total number of
proteins observed on 2-D gels when the alkylaryl aminosulphobetaine zwitterionic detergent C80 was used.
However, a number of key membrane-associated proteins could not be detected. Nevertheless, in the case of
plant plasma membranes, two of the most abundant
hydrophobic proteins (water channels and H+ATPase)
could be detected after solubilisation with C80 [60]. In
a further study on plant plasma membrane proteins,
Santoni et al. [61] purified plant plasma membranes and
were able to enrich for integral membrane proteins by further washing with sodium carbonate at pH 11 to remove
peripheral membrane proteins. Many of these proteins
could then be viewed on 2-D gels after solubilisation in a
C80-containing lysis buffer. Similar carbonate treatments
were carried out by Molloy et al. [28, 29] to extract bacterial outer membrane proteins. Carbonate treatment is
essentially an enrichment and extraction procedure
which, coupled with an adequate solubilisation procedure, increases the likelihood of low copy number membrane proteins being present on the 2-D gel map.
In addition to carbonate washing, organic solvent extraction has been carried out to enhance representation of
hydrophobic proteins from E. coli on 2-D gels [56]. This
method, however, in which lyophilised E. coli was
extracted in a 1:1 v/v solution of chloroform and methanol, and, after evaporation, resuspended in ASB14-containing solubilisation buffer, facilitated the representation
of new hydrophobic proteins from cytoplasm and periplasm. However, it failed to resolve highly hydrophobic
transmembrane proteins and in addition produced a lipid
smear on the 2-D gels. Since the same ASB14-containing
buffer was later successfully used to resolve E. coli outer
membrane proteins by Molloy et al. [28] it is likely that the
organic solvent extraction method may have contributed
to the failure to resolve highly hydrophobic proteins.
Finally, it is important to mention a recent study by Henningsen et al. [27], comparing the solubilisation abilities of
five surfactants, namely CHAPS, C80, ASB-14, ASB-16
and SB3-10. The solubilisation buffer used with all deter-
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample preparation
1415
gents in this case was 7 M urea, 2 M thiourea, 50 mM DTT,
2 mM TCEP-HCl, 0.5% carrier ampholytes pH 3–10 and
2% detergent (except in the case of SB3–10, where the
urea concentration was limited to 5 M). Henningsen et al.
[27] found that C80 appeared to be the best compound
for solubilisation of the intrinsic membrane receptor,
rP2X3, followed, to a much lesser extent, by CHAPS.
The order of detergent solubilisation potency for rP2X3
was: C80 . CHAPS . ASB14 . SB3–10 ASB16. The
authors also compared the solubilisation efficacies of
C80, CHAPS and ASB14 for the integral membrane protein FLAG-hH2R and revealed that, again, C80 was the
most potent solubiliser, but contrary to the results
obtained with rP2X3, ASB14 was more effective than
CHAPS (order of solubilisation potency: C80 . ASB14
. CHAPS) at solubilizing FLAG-hH2R. These results
demonstrate that choice of detergent for optimal solubilisation clearly depends on the nature of the protein to be
solubilised.
3.3 Prelabelling applications
The first pre-electrophoretic labelling technique described for 2-DE was traditional radioactive labelling of
E. coli proteins followed by autoradiography [12]. Since
then, prelabelling has expanded into the area of fluorescent dye technology. The first prelabelling fluorescent
dyes were monobromobimane [62, 63] and 2-methoxy2,4-diphenyl-3(2H)-furanone (MDPF). The main disadvantage of MDPF is that it reacts with primary amino groups,
thereby altering the protein’s charge and pI, analogous to
the effect of carbamylation. Monobromobimane, on the
other hand, reacts with the free sulphhydryl groups of
cysteine residues in proteins, and therefore, since SH
groups are neutral, protein charge is unaffected. In order
to generate free 2SH groups, reduction is necessary and
therefore this dye is not suitable for nonreducing IEF.
Another limitation of 2SH group labelling is that not all
proteins contain cysteines and therefore cysteine-less
proteins cannot be visualised by this method. In addition,
the intensity of labelling is not linearly dependent on the
amount of protein present in a single spot since it also
depends on how cysteine-rich the protein is. A slight disadvantage of monobromobimane, particularly concerning 2-D gels, is that its sensitivity is comparable with that
of Coomassie. An advantage of 2SH labelling is that it
functions like alkylation after reduction, blocking the
reduced sulphhydryl groups and preventing reformation
of disulphide bridges. Theoretically, therefore and in practice, pre-electrophoretic sulphhydryl labelling improves
the 2-D gel map at the basic end, where depletion of
DTT is otherwise likely to result in a streaking effect due
to protein aggregation and precipitation.
1416
M. M. Shaw and B. M. Riederer
Until recently, no 2SH binding fluorophore has surpassed
monobromobimane. It is possible, however, that a new
fluorophore, “Superbright” – currently under development
at PerkinElmer (Cambridge, UK) – will replace monobromobimane due to its increased intensity [64]. Amersham Biosciences have also manufactured a series of Cy
dyes which bind to sulphhydryl groups, which, like Superbright” are not yet commercially available.
The current commercially available Cy dyes label lysine
residues and match the charge on the lysine so these
dyes are suitable for IEF [65, 66]. The difference gel electrophoresis (DIGE) methodology from Amersham offers
three spectrally distinct CyDyes (Cy2, Cy3 and Cy5). This
enables three different samples to be run on a single gel,
which has the advantage of excluding variations in, for
example, transfer to the second dimension after focusing,
second dimension gel gradients or %T:%C ratios which
may then interfere in comparative analysis. A further
application of the fluorescent CyDyes is the inclusion of
an “internal standard” [67]; a pool of all the experimental
samples labelled with one of the CyDye DIGE fluors. The
internal standard is then run on every single gel along with
each individual sample. This means that every protein
from all samples of the experiment will be represented in
the internal standard. If a variation is observed in, for
example, a drug-treated sample when compared to the
control and the same variation is observed in the internal
standard belonging to the same gel as the drug-treated
sample but not of the control, then the apparent biological
variation observed is most likely to be an artefact due to
gel handling/first dimension transfer differences etc. and
not a true biological difference. Thus the application of an
internal standard using DIGE technology eliminates gelto-gel variation.
4 Conclusions
Progress in recent years in sample preparation methodology for 2-DE has focused on improvements in sample
prefractionation, results visualisation and sample buffer
constituents to achieve better representation of poorly
soluble proteins. Since the introduction of thiourea into
the standard sample buffer protocol, research has been
steered towards the synthesis of novel detergents for
optimal solubilisation and comparison of the effectiveness of detergents and combinations of detergents.
Current research into detergent-aided solubilisation for
poorly soluble proteins suggests that C80 is likely to be
the best surfactant, followed by ASB14 or CHAPS. In
some cases combination of CHAPS with another surfactant (e.g. NDSB256) improves solubilisation potential.
Generally, CHAPS appears to be more effective than
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Proteomics 2003, 3, 1408–1417
SB3–10 or ASB16 and CHAPS has also been found to be
more effective than the traditional nonionic detergents,
NP-40 and Triton X-100. It must be taken into account,
however, that a variety of factors influence the optimal
sample preparation protocol, some of which have been
highlighted in this review. Our goal has been to provide a
general protocol and to underline some of the reasons
why certain proteins may not be optimally resolved. It is
clear that for optimal protein representation a variety of
sample buffers may need to be tested in a trial-and-error
manner. Unfortunately, no mathematical model is available to determine the optimal sample buffer and since
the purpose of 2-DE is often to compare and analyse the
(in many cases unknown) constituents of samples, the
realisation of such a model becomes appreciable only as
the expanding proteome and 2-D gel map databases fully
unravel the protein constituents of biological tissues.
Received December 20, 2002
5 References
[1] Jackowski, G., Pielucha, K., J. Photochem. Photobiol. B
2001, 64, 45–54.
[2] Lippincott, J., Apostol, I., Anal. Biochem. 1999, 267, 57–64.
[3] Riederer, B. M., Shaw, M. M., in: Proc. Swiss Proteomics
Society, Fontis Media, Lausanne 2002, pp. 232–235.
[4] Görg, A., Obermaier, C., Boguth, G., Csordas, A. et al., Electrophoresis 1997, 18, 328–337.
[5] Molloy, M., Anal. Biochem. 2000, 280, 1–10.
[6] Rabilloud, T., Adessi, C., Giraudel, A., Lunardi, J., Electrophoresis 1997, 18, 307–316.
[7] Musante, L., Candiano, G., Ghiggeri, G. M., J. Chromatogr.
B 1998, 705, 351–356.
[8] Pasquali, C., Fialka, I., Huber, L. A., Electrophoresis 1997,
18, 2573–2581.
[9] Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I. et al.,
Electrophoresis 1998, 19, 837–844.
[10] Rabilloud, T., Electrophoresis 1998, 19, 758–760.
[11] Galvani, M., Rovatti, L., Hamdan, M., Herbert, B., Righetti,
P. G., Electrophoresis 2001, 22, 2066–2074.
[12] O’Farrell, P. H., J. Biol. Chem. 1975, 250, 4007–4021.
[13] Ames, G. F. L., Nikaido, K., Biochemistry 1976, 15, 616–623.
[14] Harder, A., Wildgruter, R., Nawrocki, A., Fey, S. J. et al., Electrophoresis 1999, 20, 826–829.
[15] Burghes, A. H. M., Dunn, M. J., Statham, H. E., Dubowitz, V.,
Electrophoresis 1982, 3, 185–196.
[16] Klose, J., Humangenetik 1975, 26, 231–243.
[17] Lenstra, J. A., Bloemendal, H., Eur. J. Biochem. 1983, 135,
413–423.
[18] Perdew, G. H., Schaup, H. W., Selivonchick, D. P., Anal. Biochem. 1983, 135, 453–455.
[19] Navarette, R., Serrano, R., Biochim. Biophys. Acta 1983,
728, 403–408.
[20] Rabilloud, T., Electrophoresis 1996, 17, 813–829.
[21] Righetti, P. G., Tudor, G., Gianazza, E., J. Biochem. Biophys.
Methods 1982, 6, 219–227.
[22] Hoving, S., Gerrits, B., Voshol, H., Müller, D. et al., Proteomics 2002, 2, 127–134.
Proteomics 2003, 3, 1408–1417
[23] Herbert, B., Galvani, M., Hamdan, M., Olivieri, E. et al., Electrophoresis 2001, 22, 2046–2057.
[24] Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J. et al.,
Electrophoresis 1998, 19, 845–851.
[25] Righetti, P. G., Gelfi, C., Bossi, M. L., Boschetti, E., Electrophoresis 1987, 8, 62–70.
[26] Rimpilainen, M. A., Righetti, P. G., Electrophoresis 1985, 6,
419–422.
[27] Henningsen, R., Gale, B. L., Straub, K. M., DeNagel, D. C.,
Proteomics 2002, 2, 1479–1488.
[28] Molloy, M. P., Phadke, N. D., Maddock, J. R., Andrews, P. C.,
Electrophoresis 2001, 22, 1686–1696.
[29] Molloy, M. P., Herbert, B. R., Slade, M. B., Rabilloud, T. et al.,
Eur. J. Biochem. 2000, 267, 2871–2881.
[30] Liberatori, S., Bini, L., De Felice, C., Magi, B. et al., Electrophoresis 1997, 18, 520–526.
[31] O’Connell, K. L., Stults, J. T., Electrophoresis 1997, 18, 349–
359.
[32] Sarto, C., Marocchi, A., Sanchez, J.-C., Giannone, D. et al.,
Electrophoresis 1997, 18, 599–604.
[33] Olivieri, E., Herbert, B., Righetti, P. G., Electrophoresis 2001,
22, 560–565.
[34] Hondermarck, H., El Yazidi-Belkoura, I., Vandermoere, F.,
Adriaenssens, E. et al., in: Proc. Swiss Proteomics Society,
Fontis Media, Lausanne 2002, pp. 41–43.
[35] Hogeboom, G. H., Methods Enzymol. 1955, 1, 16–19.
[36] Fleischer, S., Kervina, M., Methods Enzymol. 1974, 31, 6–41.
[37] Reid, E., Williamson, R., Methods Enzymol. 1974, 31, 713–
733.
[38] André, M., Servis, C., Lévy, N., Lamer, S., Felley-Bosco, E.,
in: Proc. Swiss Proteomics Society, Fontis Media, Lausanne
2002, pp. 161–162.
[39] Laemmli, U. K., Favre, M., J. Mol. Biol. 1973, 80, 575–599.
[40] Wilson, D., Hall, M. E., Stone, G. C., Rubin, R. W., Anal. Biochem. 1977, 83, 33–44.
[41] Lollo, B. A., Harvey, S., Liao, J., Stevens, A. C. et al., Electrophoresis 1999, 20, 854–859.
[42] Koll, R. A., Klinkmann, J., Richter, W. O., Artif. Organs 2002,
26, 145–151.
[43] Righetti, P. G., Chiari, M., Gelfi, C., Electrophoresis 1988, 9,
65–73.
[44] Anderson, N. G., Anderson, N. L., Tollaksen, S. L., Clin.
Chem. 1979, 25, 1199–1210.
[45] Edwards, J. J., Tollaksen, S. L., Anderson, N. G., Clin. Chem.
1982, 28, 941–948.
 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Sample preparation
1417
[46] Rabilloud, T., Valette, C., Lawrence, J., Electrophoresis
1994, 15, 1552–1558.
[47] Issaq, H. J., Conrads, T. P., Janini, G. M., Veenstra, T. D.,
Electrophoresis 2002, 23, 3048–3061.
[48] Kachman, M. K., Wang, H., Schwartz, D. R., Cho, K. R., Lubman, D. M., Anal. Chem. 2002, 74, 1779–1781.
[49] Bier, M., Egen, N. M., Allgyer, T. T., Twitty, G. E., Mosher, R.
A., in: Gross, E., Meienhofer, J. (Eds.), Peptides: Structure
and Biological Functions, Pierce Chemical Co., Rockford,
IL, 1979, pp. 79–80.
[50] Righetti, P. G., Wenisch, E., Faupel, M., J. Chromatogr. 1989,
475, 293–309.
[51] Corthals, G. L., Molloy, M. P., Herbert, B. R., Williams, K. L.,
Gooley, A. A., Electrophoresis 1997, 18, 317–323.
[52] Zou, X., Speicher, D. W., Anal. Biochem. 2000, 284, 266–
278.
[53] Zou, X., Speicher, D. W., Proteomics 2002, 2, 58–68.
[54] Michel, P., Reymond, F., Arnaud, I., Hoving, S. et al., in: Proc.
Swiss Proteomics Society, Fontis Media, Lausanne 2002,
pp. 200–201.
[55] Görg, A., Obermaier, C., Boguth, G., Weiss, W., Electrophoresis 1999, 20, 712–717.
[56] Molloy, M. P., Herbert, B. R., Williams, K. L., Gooley, A. A.,
Electrophoresis 1999, 20, 701–704.
[57] Chevallet, M., Santoni, V., Poinas, A., Rouquie, D. et al.,
Electrophoresis 1998, 19, 1901–1909.
[58] Nouwens, A. S., Cordwell, S. J., Larsen, M. R., Molloy, M. P.
et al., Electrophoresis 2000, 21, 3797–3809.
[59] Macri, J., McGee, B., Thomas, J. N., Du, P. et al., Electrophoresis 2000, 21, 1685–1693.
[60] Görg, A., Biochem. Soc. Trans. 1993, 21, 130–132.
[61] Santoni, V., Rabilloud, T., Doumas, P., Rouquié, D. et al.,
Electrophoresis 1999, 20, 705–711.
[62] Jackson, P., Urwin, V. E., Mackay, C. D., Electrophoresis
1988, 9, 330–339.
[63] Jackson, P., 1994, US Patent No. 5,320,727.
[64] Jackson, P., Kramer, S., Sissors, D., Erickson, T. et al., in:
Proc. Swiss Proteomics Society, Fontis Media, Lausanne
2002, pp. 55–58.
[65] Minden, J., Waggoner, A., 2000, US Patent No. 6,127,134.
[66] Unlu, M., Morgan, M. E., Minden, J. S., Electrophoresis
1997, 18, 2071–2077.
[67] Driver, S., Ghouze, F., Alban, A., Orange, P., Currie, I., in:
Proc. Swiss Proteomics Society, Fontis Media, Lausanne
2002, pp. 53–54.