2-D Electrop horesis

A Comparison of Carrier Ampholyte and Immobilized pH Gradients

Creating a carrier ampholyte Creating an immobilized pH gradient

pH gradient An immobilized pH gradient (IPG) is created and bisacrylamide monomers to form a
by covalently incorporating a gradient of buffers polyacrylamide gel. Thus the pH at any point in
into a polyacrylamide gel at the time it is cast. the gel is determined by the mixture of buffering
Immobiline® acrylamido buffers are a set of groups in the neighborhood.
simple molecules, each with a single acidic or The IPG gel casting process (A), (B), (C) is
basic buffering group linked to an acrylamide similar to casting an acrylamide gradient gel.
monomer. The acrylamide portion of the buffer Essentially it uses a two-chamber gradient maker
readily co-polymerizes with normal acrylamide with primarily acidic acrylamido buffers in one
chamber and primarily basic buffers in the other.
The concentrations of the various buffers in the
two chambers define the range and shape of
the pH gradient produced.
For improved performance and simplified
handling, the gel is cast onto a plastic backing,
then washed (D) to remove
catalysts and unpolymerized
monomers, and dried (E).
The particular mixture of
immobilized buffer (F) at
any given point in the gel
determines the local pH.
For use as the IEF dimension
of 2-D electrophoresis, the
dried gel is cut into strips
Carrier ampholytes are a heterogeneous mixture of synthetic polymers incorporating (G). Produced in large
a variety of both acidic and basic buffering groups. Ampholyte molecules have net batches, the IPG gel strips
charges that depend on the pH of the environment and the number and pKs of offer convenience and
the particular mixture of acidic and basic groups on the particular molecule. For reproducibility not easily
isoelectric focusing (IEF), carrier ampholytes are incorporated into gels as a compo- achieved with lab-cast gels.
nent of the gel casting solution. In the absence of an electrical field (A), the carrier
ampholytes are randomly distributed and establish a uniform pH throughout the
gel matrix — about pH 7 when creating a pH 3 –10 gradient.
When an electrical field is applied (B), usually through an acid electrode solution at
the anode (+) and a basic electrode solution at the cathode (–), all carrier ampholytes
with a net charge will start to migrate: those with a net negative charge and low pI
value move toward the anode, those with a net positive charge and a high pI value
move toward the cathode, and those with no net charge (neutral) do not move.
The ampholytes with the more extreme pI values can migrate closer to the appro-
priate electrode solution before they are titrated to the pH equal to their pI. Thus
the pH gradient is established by the mobile carrier ampholytes (C). At equilib-
rium, the pH at any point in the gel is determined by the average pI of the soluble
carrier ampholytes at that point.

Time-dependent distortion of
carrier ampholyte pH gradients
With carrier ampholytes in polyacrylamide gels, the pH gradient never reaches a true
stationary equilibrium: over time there is a significant drift of the pH gradient toward
the cathode (–). This is primarily due to electroendosmotic flow, the electrically driven
movement of water. The result is that separated proteins do not remain focused at a
specific point in a gel, but move in a time-dependent way, usually toward the cathode.
Stability of immobilized pH gradients
An example of cathodic drift is shown in Figures 1 and 2, below. Over time the immobilized pH gradient is stable. Once the proteins reach their pI point (Figures 3 and 4),
Another source of distortion of carrier ampholyte pH gradients arises from they remain focused at a specific position and do not drift toward either electrode. Focusing conditions
handling the tube gels commonly used for the IEF dimension of 2-D electrophoresis. must provide a stable temperature and prevent exposure to atmospheric CO2.
After being removed from the glass tube holder, the long, thin gels can be easily IPG gel strips with a plastic backing are not subject to physical distortion or breaking during handling,
stretched or broken as they are applied to the second dimension gel. which also contributes to the improved simplicity and reproducibility of 2-D electrophoresis.

Figure 1. Time course of IEF with Figure 3. Time course of

carrier ampholytes. Marker proteins IEF on an immobilized pH
(soybean trypsin inhibitor, pI 4.6; gradient gel. Bean seed
lactoglobulin, pI 5.3; conalbumin, pI protein extracts (Vicia faba)
5.9) were focused for the indicated were separated on an
time in a pH 4-6 carrier ampholyte immobilized linear pH 4-8
mix on a flatbed acrylamide gel. gradient flatbed gel for the
[From Görg, et al, in Two-Dimensional indicated time. [From Görg,
Electrophoresis, Endler, A.T. and et al, in Two-Dimensional
Hanash, S. eds, VCH-Verlag, Electrophoresis, Endler, A.T.
Weinheim 1989. With permission and Hanash, S. eds, VCH-
of the author.] Verlag, Weinheim 1989. With
permission of the author.]

2h 3.5h 4h 5h 3h 6h 9h 12h

Figure 2. Shape of Figure 4. Shape of

carrier ampholyte pH immobilized pH
gradient over time. gradients over time.
Curves plotted from Apparent pH curves
position of pI markers are plotted from
at indicated time in position of selected
Figure 1. bands from Figure 3.

80-6419-53 Rev A / 3-98 Copyright © 1998 Amersham Pharmacia Biotech