The most common problem encountered with 2-D
gels is protein streaking, whether in the horizontal
(Figure 1) or vertical (Figure 2) direction. This
streaking effect can be due to a number of different
factors, but, in keeping with the logic of this technique,
generally horizontal streaks are due to problems
with isoelectric focusing, while vertical streaks
are due to poor protein separation during sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). This article focuses on the most common
causes of both horizontal and vertical streaking
on gels, and offers suggestions to prevent them.
Figure 2 - Examples of vertical streaking. a) Approximately 300 μg of
E. coli protein loaded onto a 17-cm pH 3–10 ReadyStrip™ IPG strip (Bio-Rad). b) A prefractionated sample of Pseudomonas putida loaded onto a
17-cm pH 3–10 ReadyStrip IPG strip. Gel image was annotated using The
Discovery Series™ PDQuest™ 2-D analysis software (Bio-Rad).
Figure 1 - Example of horizontal streaking and its prevention. Detergent was
effectively removed by the ReadyPrep™ 2-D cleanup kit (catalog #163-2130, Bio-Rad Laboratories, Hercules, CA), leading to improved 2-D SDS-PAGE separation.
E. coli extracts were spiked with 1% SDS. a) Untreated sample. b) Sample
treated with the ReadyPrep 2-D cleanup kit.
Causes of horizontal
streaking
Incomplete or excessive
isoelectric focusing
All protein samples are different and require different
strategies for good isoelectric focusing; consequently,
finding the optimal focusing conditions is necessary.
Suggested focusing protocols for the various immobilized
pH gradient (IPG) strip lengths and pH ranges
are supplied with ReadyStrip IPG strips. These make
a good starting point for optimization. However,
incomplete focusing or overfocusing is always a possibility,
and both can lead to horizontal streaking.
As would be expected, incomplete focusing results in
horizontal streaks, since the proteins have not had
the opportunity to be focused into discrete spots.
Often overlooked as a potential explanation
for incomplete focusing is the focusing of different
samples, different pH range IPG strips,
or both at the same time. Conventional IEF
cells set a total current limit for the whole tray;
thus, if one particular sample is more conductive,
it will draw most of the current and hence
decrease the focusing rate of the other strips in
the tray. Therefore, samples with very different
conductivities should be run separately.
In general, overfocusing is not as much of a
problem as underfocusing. Overfocusing is not
an issue up to about 100,000 V/hr, but above
this level, the potential for electroosmosis can
result in water and protein movement in the
IPG strip and horizontal streaks.
Protein overloading
The total amount of protein to load onto an
IPG strip usually depends on the length of the
strip and the stain that will be used to visualize
the results (see Table 1). Certain circumstances
require additional consideration, such as when
particular proteins constitute a large percentage
of the total protein. This is the case with serum
albumin, which makes up 70–90% of total
serum protein. Loading the recommended
amount of protein likely would obscure many
other proteins, because they would be masked
by albumin. Under these circumstances, users
should resist compensating by loading more
protein—this could worsen the horizontal
streaking. Instead, the serum albumin can be
removed using Aurum™ Affi-Gel® Blue mini columns (catalog
#732-6708, Bio-Rad) and then
the recommended amount of
protein loaded.
High protein loads, which result from
loading too much protein into sample
cups during cup loading, or from using
IPG strips for preparative runs, can
allow protein aggregation. This may
result in protein precipitation at a particular
protein’s isoelectric point (pI
fallout), which leads to horizontal
streaking. A slight increase in the IPG
strip rehydration volume (by ~10%)
may result in improved focusing. The
suggestions described below for using
reduction–alkylation and stronger
detergents can also help to reduce protein–protein interactions and thus
some of these problems.
Sample preparation
problems
The most common cause of horizontal
streaking is a problem with sample
preparation. Any contaminant with a
net ionic charge will affect isoelectric
focusing and lead to horizontal streaking.1 Common contaminants include
salt, detergents (see Figure 1), peptides,
nucleic acids, lipids, and phenolic compounds.
Ionic contaminants usually cause high current
(around 50 μA) during isoelectric focusing. This current
will limit the voltage of the isoelectric focusing
cell and increase isoelectric focusing run times, since
the voltage will not increase until the ionic contaminants
have been depleted, a process that takes many
hours.2 This generally leads to underfocusing and
streaking. Ionic contaminants can also cause excessive
IPG strip heating or strip burning. To alleviate
these problems, salt can be removed from the sample
using dialysis, desalting columns, or the ReadyPrep
2-D cleanup kit, which removes salts and most other
ionic contaminants. Certain charged detergents,
such as SDS, can coat proteins and drastically alter
their net charge, ultimately affecting their isoelectric
point and leading to streaking and loss of sample
from the IPG strip.
Nucleic acid contamination of samples can also
contribute to horizontal streaking.3 Many proteins
bind nucleic acids, which can generate a
mixed population of protein–nucleic acid
species, each with distinct isoelectric points.
Nucleic acids can be removed from samples by
treating the samples with nucleases prior to IEF.4
However, these nucleases will be present in the
2-D gel. Alternatively, nucleic acids can be removed by ultracentrifugation in the presence
of spermine.5
Polysaccharides that contain sialic acid and are negatively
charged can produce horizontal streaking
artifacts similar to those seen with nucleic acids.
Uncharged polysaccharides can also cause streaking
by blocking the pores of the gel, preventing sample
entry into the IPG strip, and physically impeding
focusing. Ultracentrifugation is often sufficient to
remove carbohydrates.
In addition, carbamylation of polypeptides can generate
charge heterogeneity in individual proteins, and
although discrete trains of spots are often formed,
under certain conditions—such as when broad pH
gradients are used—smearing between spots can
occur. Protein samples should be prepared using high-purity
urea and should not be heated above 30 °C.
The last cause of horizontal streaking related to sample
preparation is disulfide bond formation.6 When
disulfide bonds occur in a protein sample prior to 2-D, they occur randomly intra- and intermolecularly.
Disulfide bond formation creates various protein
configurations, including minor isoelectric point
variants (appearing on a 2-D gel as a widening of a
single protein spot) and multimers of the same protein
(appearing as streaking tails on spots, phantom
spots, missing spots, and smearing). Preventing disulfide
bond formation eliminates
these artifacts. Disulfide bond artifacts
are particularly problematic in
two instances: 1) analysis of basic
proteins, and 2) use of longer IPG
strips. Horizontal streaking due to
disulfide bond formation can be
reduced using the ReadyPrep reduction–alkylation kit (catalog #163-2090, Bio-Rad) in conjunction with
cup loading of IPG strips.7
Poor protein
solubilization
Failure to completely solubilize all
the proteins in a sample will result
in horizontal streaking. The main
components of standard sample
buffers used in 2-D electrophoresis
to maintain sample solubility are
urea, a nonionic detergent such as
Nonidet P-40 (NP-40) (Shell
International Petroleum Co.,
Houston, TX) or a zwitterionic
detergent such as 3-[3-cholamidopropyl)
dimethyl-ammonio]-1-propanesulfonate (CHAPS), and a
reducing agent such as dithiothreitol
(DTT). Other compounds can
be included in this mixture, for
example, thiourea,8 or novel detergents,
especially those in the amido
sulfobetaine family, such as amidosulfobetaine-14 (ASB-14),5 to
improve the solubilization of membrane
proteins. The combined
effect of these reagents is to reduce
horizontal streaking by better solvating
the hydrophobic regions on
the proteins and diminishing aggregation
due either to hydrogen
bonding among proteins or to interactions
with the IPG gel matrix.
Finally, it is always good practice to
remove insoluble protein complexes
by centrifugation prior to
isoelectric focusing to prevent them
from interfering with the entry of
other proteins into the gel matrix.
Electroendoosmotic flow
Mainly a problem during isoelectric focusing of basic
proteins, electroendoosmotic flow can contribute to
horizontal streaking due to water transport from the
cathode to the anode. The water flow slows protein
migration in the opposite direction and can also
cause strip dehydration at the cathode end.
Solutions to this problem include replacing the
paper wick at the cathode with a fresh water-soaked
wick or adding organic modifiers such as glycerol,
isopropyl alcohol, or methylcellulose to the rehydration/sample solution.3
Causes of vertical
streaking
Poor protein solubility
Following the first-dimension separation, all of the
proteins have moved to their isoelectric points and
carry no net charge. The ionic strength of the
medium is also very low, because small ions have all
migrated out of the first-dimension strip. Proteins
generally have minimal solubility under these conditions
and are often precipitated within the gel matrix
despite the presence of detergents, chaotropic agents,
and other solubilizing additives. The major purpose of
the equilibration step following the first dimension is
to coat the proteins with SDS, giving them a negative
charge and making them soluble again so that
they may enter the second-dimension gel.
Protein overloading
When a large quantity of protein is loaded, or when
the sample contains proteins of particularly high
abundance, resolubilization during equilibration may
be incomplete, resulting in vertical streaking and
tailing of the most intense protein spots. This can be
prevented by limiting the amount of protein loaded
onto the first-dimension strip. It is often possible to
compensate for a lower protein load by using a more
sensitive staining technique (for example, silver
stain, catalog #161-0449 [Bio-Rad] or SYPRO Ruby
protein gel stain [Invitrogen, Carlsbad, CA], catalog
#170-3125, instead of Coomassie blue stain [BASF
Aktiengesellschaft, Berlin, Germany]). In some
cases, vertical streaking of abundant proteins can be
prevented by prolonging the equilibration time (see
“Ineffective equilibration,” below).
Overfocusing
Isoelectric precipitation increases with focusing
time; therefore, vertical streaking may be minimized
by ensuring that first-dimension isoelectric focusing
is not conducted for any longer than necessary.
Ineffective equilibration
Equilibration should be carried out in a manner that
guarantees good penetration of SDS and hence protein
solubilization. The equilibration tray should be
shaken or rocked to ensure continual movement of
solution. Equilibration may be prolonged as long as
45 min if necessary. This may result in the loss of
some small polypeptides (<15 kD), but loss of larger
proteins is generally insignificant even with prolonged
equilibration. SDS should be present at a
concentration of at least 2% (wt/vol), particularly if
high (>2%) concentrations of detergent were used in
the first dimension. Other components of the equilibration
solution are important as well. The solution
should be buffered, particularly if an alkylation step
is employed, because iodoacetamide treatment generates acid. The optimal pH for equilibration is 8.8.
Glycerol, which should be present at a concentration
of at least 20% (vol/vol), is a particularly important
component of the equilibration solution, and its
omission will result in vertical streaking.
Protein oxidation
Oxidative crosslinking or protein refolding should
be prevented during all steps of the 2-D process;
protein oxidation during the second-dimension
separation can result in vertical streaking.
Treatment with the ReadyPrep reduction–alkylation
kit prior to first-dimension focusing can block
cysteine sulfhydryl groups and prevent their reoxidation.
If the sample is not alkylated, alkylation
should be performed in a second equilibration step
with 2.5% iodoacetamide. If alkylation is not
desired, a sufficient quantity of DTT, at least 1%,
should be present in the equilibration solution. The
DTT should be added to the equilibration solution
immediately before use, since it may oxidize if
stored. DTT is negatively charged under second-dimension
conditions and will migrate ahead of
proteins in the gel, ensuring a reducing environment
during the separation.
Poor reagent quality or
improperly prepared solutions
Vertical streaking and other second-dimension
problems often result from poor reagent quality or
from mistakes in solution preparation. Care should
be taken to use the highest-quality reagents (for
example, ReadyPrep proteomics grade water, catalog
#163-2091 [Bio-Rad]), and the pH of all buffers
should be verified. Precast second-dimension gels
should be used before their expiration date. Poor-quality
acrylamide or old acrylamide stock solutions
can cause vertical streaking due to an incompletely
polymerized second-dimension gel. Reusing electrophoresis running buffer can result in poor
separation and vertical streaking due to the depletion
of ions and SDS in the running buffer. This
practice should be avoided if vertical streaking is a
persistent problem.
Poor placement of the IPG
strip on the second-dimension
gel
Vertical streaking can also be the result of gaps
between the IPG strip and the second-dimension gel
or damage to the IPG strip during application. The
second-dimension gel should have a straight, level
top edge, and care should be taken that the IPG strip
is in direct contact with the second-dimension gel
along its entire length. The IPG strip should not be
twisted during placement. This can be prevented by
ensuring that the plastic backing of the IPG strip is
pressed firmly against one of the second-dimension
gel plates. An agarose overlay should be used to prevent
the IPG strip from coming loose or moving.
Bubbles in the agarose overlay should be minimized.
Uneven pore size in the IPG
strip following isoelectric
focusing
After the first-dimension separation, there are often
variations in the thickness of the IPG strip. This
swelling or thinning of some sections is caused by
water movement in the gel.1 In thinner sections of
the IPG strip, the gel pore size may have been
reduced to the point that protein migration out of
the IPG strips will be slowed or prevented. This can
result in vertical streaks or, in severe cases, complete
protein loss from trapping within the IPG strip.
Water movement during first-dimension separation
can be minimized by ensuring that the sample is well
desalted and that focusing is not carried out any
longer than necessary.
Vertical gaps or vertical blank
stripes
Vertical gaps or blank stripes, for the purposes of this
discussion, are different from vertical streaking.
These problems can be due to trapped air bubbles in
the agarose overlay on the IPG strip, excessive DTT
(>50 mM) in the IPG sample buffer, or a compromised
IPG strip (either insufficiently rehydrated or
torn during handling). Blank stripes near the electrode,
especially the cathode, can be caused by a
buildup of salt.
Summary
As alluded to at the beginning of this paper, streaking
on 2-D gels is a common and complex problem.
The authors have identified useful remedies for some
of the main causes of such streaking, although they
have not presented an exhaustive list of the potential
causes. In addition, the great deal of variation in
the characteristics of protein samples means that
each sample will behave uniquely. With such variety
in the composition of protein samples, the sample
preparation step becomes important to prevent frequently
observed streaking patterns; this application
note has focused on a variety of approaches to help
achieve this.
References
- Rabilloud T. Proteome research: two-dimensional gel
electrophoresis and identification methods. Berlin:
Springer, 2000.
- Rémy A. Focusing strategy and influence of conductivity
on isoelectric focusing in immobilized pH gradients. Bio-Rad bulletin 2778, 2000.
- Görg A, Obermaier C, Boguth G, Csordas A, Diaz JJ,
Madjar JJ. Very alkaline immobilized pH gradients for
two-dimensional electrophoresis of ribosomal and nuclear
proteins. Electrophoresis 1997; 18:328–37.
- Garfin D, Heerdt L, eds. 2-D electrophoresis for proteomics:
a methods and product manual. Bio-Rad bulletin
2651, 2000.
- Rabilloud T. Solubilization of proteins for electrophoretic
analyses. Electrophoresis 1996; 17:813–29.
- Walsh BJ, Herbert B. Setting up two-dimensional gel electrophoresis
for proteome projects. ABRF News 1998;
9:11–21.
- Bio-Rad Laboratories. Cup loading tray for the PROTEAN
IEF cell instruction manual. Bio-Rad bulletin
4006216, 2000.
- Rabilloud T. Use of thiourea to increase the solubility of
membrane proteins in two-dimensional electrophoresis.
Electrophoresis 1998; 19:758–60.
Dr. Berkelman is Senior Staff Scientist; Ms. Brubacher is Senior
Product Manager; Dr. Chang is no longer with the company, but
during her tenure was a Senior Technical Support Specialist focused
in the area of proteomics; Dr. Cross is Northern European Product
Manager for Protein Technologies; and Dr. Strong is R&D Group
Leader, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Dr.,
Hercules, CA 94547, U.S.A.; tel.: 510-741-1000; fax: 510-741-5800; home page: www.expressionproteomics.com.