Application Note:

Capillary Isoelectric
Focusing of a Complex
Protein Mixture

No. 5.200405
Rev. A
2004 Target Discovery, Inc.

Target Discovery, Inc.

4015 Fabian Way
Palo Alto, CA 94303
Tel: 650-812-8100
www.targetdiscovery.com/eotrol

Application Note:
Application Challenge:
Analyte:
Product Family:
Product Used:

No. 5.200404
Basic (> pH 10) Protein Focusing
E. coli Extract
EOTrol Dynamic Coatings
EOTrol LN and EOTrol HR

CAPILLARY ISOELECTRIC FOCUSING OF A COMPLEX PROTEIN MIXTURE

William W. P. Chang, David C. Bomberger, Luke V. Schneider
Target Discovery, Inc. Palo Alto, CA 94303 USA
Capillary isoelectric focusing (CIEF) is a high-resolution technique for protein separation
based on differences in isoelectric points (pI). In contrast to gel-based IEF, CIEF is rapid and
easily automated. The separation of proteins with
pI as small as 0.004 has been
demonstrated1. The separation power of CIEF is estimated to be ~1000 peaks in a 65 cm
capillary2. However, the attainment of high resolution and reproducibility in CIEF requires that
the electroosmotic flow (EOF) be suppressed. Otherwise the bulk flow towards the cathode as
a result of EOF will prevent/destroy the focusing of individual proteins. To control EOF, many
have resorted to the use of covalently coated capillaries. Commercial coated capillaries
typically have inconsistent coating efficiency, which leads to poor reproducibility between lots
and within a lot3, and causes resolution loss due to internal ion eddies. We applied a dynamic
coating, EOTrol LN, to control EOF during CIEF separation and have achieved better
resolution than a commercial polyethylene oxide (PEO) coated capillary. EOTrol LN helped
resolved proteins with pI from 3.8 to 9.6, but higher pI proteins were eluted from the capillary
during the focusing period. When another EOTrol formulation (EOTrol HR) was applied, we
observed increased capacity of CIEF for basic proteins, with resolution up to a pI of at least 10.7
(cytochrome c), but the reversed EOF caused proteins with pI below 5.1 to be eluted from the
capillary during the focusing period. This means that two runs, one with LN and the other with
HR, can be used to provide complete CIEF resolution.
Proteins were visualized by using the CIEF as the first step in a 2 dimensional
separation scheme. Proteins in a sample were allowed to reach their pI's in a capillary operated
with a sodium hydroxide solution as the catholyte and phosphoric acid as the anolye. Although
normal practice would be to use pressure to mobilize the proteins from the capillary for
subsequent analysis after focusing, the pressure mobilization causes mixing and band
widening. An alternate approach was utilized. The capillary was cut into 2 cm sections and the
contents of each section were analyzed by gel electrophoresis. The process is shown
schematically in Figure 1. More details of the experimental procedures are contained in the
Experimental Section.

2004 Target Discovery, Inc. All rights reserved

Rev. A

Figure 1. The results of

CIEF focusing inside the
capillaries are displayed by
silver stained minigels.
After CIEF (1) is done, the
capillary is removed from
the cartridge and cut into
2-cm pieces (2). The
contents of the capillary
segments are analyzed by
size separation on
polyacrylamide gels (3).
The composite gel image
(4) demonstrates the
distribution of proteins by
their isoelectric points.

Figure 2. CIEF protein

separation profile in a
PEO-coated capillary.
The residual EOF in a
capillary covalently coated
with polyethylene oxide is
~2 to 3 x 10-9 m2V-1s-1
(data not shown). This
level of EOF can still lead
to the drainage of basic
proteins as shown by the
composite gel image.
Proteins having pI
7.6
were lost from the system.
The blank space below pI
3.8 suggests that more
acidic proteins may be
resolved, perhaps down
to the pH of the anolyte.
See the Experimental
Section for details of the
sample and instrument
setup.
2004 Target Discovery, Inc. All rights reserved

Rev. A

 2004 Target Discovery, Inc. All rights reserved

Rev. A

Figure 4. CIEF protein separation profile in a bare

silica capillary with EOTrol HR dynamic coating.
EOTrol HR produces a reversed (anodal) EOF of
approximately 2 x 10-8 (m2V-1s-1). Proteins with pI
up to 10.7 were retained in the capillary at the end of
the run. However, the trade-off is that some of the
acidic proteins (pI <5.1) were lost, flushed out by the
anodal EOF. The relatively blank space beyond the
pI 10.7 marker suggests more basic proteins can be
potentially resolved. We empirically determined that
the lysozyme marker included in the sample has an
apparent molecular weight of 12 kDa which is the
same as that of the cytochrome c (data not shown.)
It is possible that lysozyme was present, trailing the
cytochrome c, in which case this method may
potentially resolve proteins with pI values to at least
pI 11.4.

Figure 3. CIEF protein separation profile in a bare

silica capillary with EOTrol LN dynamic coating.
EOTrol LN, produces a cathodal EOF to ~2 x 10-9
m2V-1s-1. Proteins with pI up to 9.6 were retained in
the capillary at the end of the run. The resolution of
E. coli proteins (left) is comparable, if not slightly
better than that found in the PEO-coated capillary
(Fig. 2). See the Experimental Section below for
details of the sample and instrument setup.

Lower Cost and Superior Reproducibility

EOTrol LN provided comparable, if not slightly better CIEF resolution than that found
in PEO-coated capillaries, and it did it in the cheaper bare silica capillary. As shown in our
previous work3, EOTrol coated capillaries provide superior run-to-run reproducibility compared
to commercially PEO-coated capillaries, which tend to have high lot-to-lot and intra-lot
variability. EOTrol HR can be used to extend the CIEF analysis to more basic proteins.

EXPERIMENTAL
Capillaries Used in CIEF
A bare silica capillary was preconditioned by rinses with methanol, HCl and NaOH using
water rinses in between. The PEO-coated capillary was only rinsed with methanol and water to
prevent base etching of the coating. Dimensions of the capillaries: 100 m i.d., 60.2 cm in
overall length (50 cm to detector).
Sample Preparation
An E. coli stock solution was prepared by dissolving 2 mg of lyophilized E. coli extract in
0.5 mL of 1% SDS and 150 mM DTT. Proteins in the stock solution were heated at 95 C for 5
min and stored in the freezer until use. The E. coli extract (Proteomic Standard, E. coli) was
obtained from Geno Technology, Inc. (St. Louis, MO). A sample mixture of 0.8 mg/mL E. coli
stock solution was prepared in 8 M urea, 1% w/v DTT, 4% CHAPS, and 5% v/v Pharmalyte 310. pI markers were added to the sample: amyloglucosidase (70 kD & 89 kD, pI 3.8), ovalbumin
(45 kD, pI 5.1), carbonic anhydrase (29 kD, pI 7.0), myoglobin (17 kD, pI 7.6), ribonuclease A
(13.7 kD, pI 9.6), cytochrome c (12 kD, pI 10.7) and lysozyme (14.3 kD, pI 11.4). EOTrol was
added to the sample to a final concentration of 0.15% (w/v).
CE Instrument and Conditions
A Beckman-Coulter P/ACE MDQ Capillary Electrophoresis System (Beckman-Coulter
Instruments, Inc., Fullerton, CA) was used in all CIEF experiments. Experiments were carried
out with liquid cooling to maintain a run temperature of 25 C. The capillary was rinsed and
filled with the E. coli extract (see Sample Preparation above) by pressure. Separation was
carried out by applying a voltage of 25 kV between 10 mM H3PO4 and 20 mM NaOH for 130
min.
Display of CIEF Results by SDS-PAGE
At the end of the CIEF separation, the capillary was cut up into 2-cm segments. The
content of each segment was analyzed using SDS-Polyacrylamide Gel Electrophoresis (12%
Bis-Tris, Invitrogen, Carlsbad, CA). Protein bands were visualized by silver staining
(SilverXpress Silver Stain kit, Invitrogen), and images of the individual gels were integrated to
show the composites illustrated in the Figures. The complete operation is illustrated in Figure 1.

REFERENCES
[1] Shen Y et al., Anal Chem, 71:5348 (1999).
[2] Shen Y et al., Anal Chem, 72:2154 (2000).
[3] Chang W et al., Am Lab News, 36:8 (2004).

2004 Target Discovery, Inc. All rights reserved

Rev. A