Innovations Forum: Improved protein separation
The use of 24 cm, pH 3–11 NL
Immobiline DryStrip gels improves
protein separation
L. Coquet*, P. Cosette, and T. Jouenne
UMR 6522 CNRS, Proteomic Platform of the European Institute for Peptide Research,
(IFRMP 23), University of Rouen, Mont-Saint-Aignan, France
* e-mail : [email protected]
In this study, new 24 cm, pH 3–11 NL, immobilized pH gradient (IPG) gels were used to
separate a total protein extract of rat pheochromocytome cells. Results show that the
new Immobiline™ IPG strip improves separation of both acidic and basic proteins as
compared with the 18 cm, pH 3–10 NL IPG strip. This enhanced focalization was also
confirmed with results obtained for a purified basic protein (IL-7, pI = 8.9).
Introduction
The use of IPG gels (1) has enabled great progress in proteomics
by introducing greater reproducibility and resolution in 2-D gels.
However, even if 2-D electrophoresis (2DE) remains at the cutting
edge of protein separation science, some important limitations still
remain. Low-copy proteins, hydrophobic proteins, or basic proteins
are often missing (2). The visualization of basic proteins is difficult due
to inadequate isoelectric focusing resulting from the reverse cathode
to anode electro-osmotic flow. This phenomenon causes horizontal
streaking and poor resolution. Improved resolution in the alkaline area
of the gel constitutes one of the main challenges in the field of 2DE.
In this study, we have compared the capacity of the IPG strip
Immobiline DryStrip pH 3–11 NL, 24 cm, to separate proteins as
compared with that of the Immobiline DryStrip pH 3–10 NL, 18 cm.
The study was performed by using protein extracts of rat pheochromocytome cells and a recombinant human interleukin (IL-7).
Materials and methods
Sample preparation
Total proteins extracted from rat PC12 tumoral cells and recombinant
human IL-7 expressed and extracted from E. coli were lyophilized and
diluted with DeStreak Rehydration Solution (Amersham Biosciences)
containing 2% (v/v) IPG Buffer pH 3–11 NL (Amersham Biosciences).
Protein amounts were evaluated using a protein assay and measuring
the absorbance at 595 nm.
2-D electrophoresis
Protein samples (50 µg of IL-7 or 400 µg of PC12 cell protein extract)
were brought up to 400 µl with DeStreak Rehydration Solution and
2% IPG Buffer pH 3–11 NL. Samples were applied by passive
rehydration using Immobiline DryStrip Reswelling Tray (overnight for
18 cm strips; 2 days for 24 cm strips).
12 Life Science News 18, 2004 Amersham Biosciences
First-dimension electrophoresis was performed on Multiphor™ II IEF
System (Amersham Biosciences). For Immobiline DryStrip pH 3–10 NL,
18 cm, focusing conditions were 150 V for 1 h, 350 V for 15 min, 750 V
for 45 min, 1500 V for 1 h and 3500 V for 17 h (1 mA, constant) for a
total of 61.8 kVh.
For Immobiline DryStrip pH 3–11 NL, 24 cm, the focusing conditions
were: 200 V for 1 h, 500 V for 1 h, 1000 V for 45 min, 2000 V for 45
min and 3500 V for 28 h (1 mA, constant) for a total of 100.95 kVh.
The equilibration solution contained 6 M urea, 30% (v/v) glycerol
and 2% (w/v) SDS in 50 mM Tris-HCl, pH 6.8. The first step of
equilibration (reduction) was carried out with 2% (w/v) DTT and
the second step (aklylation) with 2.5% (w/v) iodoacetamide and
0.03% (w/v) Coomassie™ Brilliant Blue R-250 (7). Both steps were
performed for 10 min each at room temperature.
The second-dimension SDS-PAGE separation was performed using a
12.5% (w/v) polyacrylamide resolving gel. For 18 cm IPG strips,
separation conditions were 10 mA/gel for 45 min, 20 mA/gel until
Coomassie Brilliant Blue tracking dye had migrated off the lower end
of the gel. For 24 cm IPG strips, the separation conditions were 15
mA/gel for 80 min and then 40 mA/gel. After migration, proteins were
visualized by colloidal blue staining.
Gel analysis
Gels were scanned using a densitometer and analyzed using
ImageMaster™ 2D Platinum software (version 5.0,
Amersham Biosciences).
Results and discussion
A new IPG gel with a slightly increased pH gradient (Immobiline
DryStrip pH 3–11 NL, 24 cm) was compared with a shorter, and
narrower pH range strip (Immobiline DryStrip pH 3–10 NL, 18 cm).
Innovations Forum: Improved protein separation
The protein pattern obtained for the PC12 protein extract indicates
that the new strip offers a good resolution over the quasi-entire pH
range. When comparing with the other strip, approximately 25% more
spots were detected (938 as compared with 746 with the
18 cm IPG). This effect is particularly remarkable in the acidic and
alkaline areas with an increase in spot numbers of 47% and 51%
respectively, as compared with 10% in the neutral zone (Table 1).
A
3
4
pH
6
5
7
8
9
10
Fig 1. 2-D gels performed
with 400 µg of PC12 cell
proteins. (A) IEF performed
using Immobiline DryStrip
pH 3–10 NL, 18 cm.
(B) IEF performed using
Immobiline DryStrip
pH 3–11 NL, 24 cm.
Colloidal blue staining.
Vertical black dotted lines
delimit the pH zones 3–5,
5–8 and 8–10/11) which
were used for spot
detection. (C) Black and
red boxes, with plain or
dotted lines, correspond to
magnification views of the
acidic and basic regions
indicated in A and B.
Table 1. Number of detected spots within pH ranges as shown in Fig 1.
pH range
4
5
pH
6
7
8
9
10
3–5
5–8
8–10/11
Total
18 cm, pH 3–10 NL
71
608
67
746
24 cm, pH 3–11 NL
132
677
129
938
This observation is not surprising and confirms data reported by
Poland (3) who attributed this effect to a better focalization in a
larger pH gradient. In addition, it might be due to the intrinsic
property of this new strip because new spots appeared in both acidic
and basic parts of the pH gradient (see magnification views in Fig 1).
To further evaluate the focusing ability of Immobiline DryStrip
pH 3–11 NL, 24 cm, a low molecular weight basic protein was
loaded at a high concentration (Fig 2). In this case, the focalization is
remarkable as compared with patterns obtained on other IPG strips
where streaking is predominant (data not shown). With this new strip,
streaking is greatly reduced and a pI of 9.2 can be estimated for IL-7
(theoretical value: 8.9).
3
B
3
IPG strip
4
5
pH
6
7
8
11
9 10 11
Fig 2. 2-D gel of
interleukin-7 (50 µg)
performed with
Immobiline DryStrip pH
3–11 NL, 24 cm. Colloidal
blue staining.
Conclusions
The present study demonstrates that Immobiline DryStrip pH 3–11 NL,
24 cm is well adapted for proteomic purposes. This new IPG strip
offers a higher resolution in the acidic and alkaline gel areas, zones in
which streaking and poor resolution generally occur.
References
1. Bjellqvist, B. et al. Isoelectric focusing in immobilized pH gradients :
principle, methodology and some applications. J Biochem Biophys Methods 6,
317–319 (1982).
2. Harry, J. L. et al. Proteomics : Capacity versus Utility. Electrophoresis 21,
1071–1081 (2000).
3. Poland, J. et al. Isoelectric focusing in long immobilized pH gradient gels to
improve protein separation in proteomic analysis. Electrophoresis 24,
1271–1275 (2003).
Ordering Information
C
Immobiline DryStrip pH 3–11 NL, 24 cm (12)
17-6003-77
Immobiline DryStrip pH 3–10 NL, 18 cm (12)
17-1235-01
Destreak Rehydratation Solution (5 × 3 ml)
17-6003-19
DeStreak Reagent (1 ml)
17-6003-18
IPG buffer 3–11 NL (1 ml)
17-6004-40
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Life Science News 18, 2004 Amersham Biosciences 13