Protein Protocols Protein Protocols

Casting IPGs 169
23
Casting Immobilized pH Gradients (IPGs)
Elisabetta Gianazza
1. Introduction
One of the main requirements for a 2-D protocol is reproducibility of spot position,
and, indeed, the technique of isoelectric focusing on immobilized pH gradients (IPGs)
is ideally suited to provide highly reproducible 1-D separations. IPGs are obtained
through the copolymerization of acidic and basic acrylamido derivatives of different
pKs within a polyacrylamide matrix (1,2) (Fig. 1). The pH gradient may be devised
by computer modeling either with a linear or with an exponential course. IPGs are cast
from two limiting solutions containing the buffering chemicals at concentrations adjusted
to give the required pH course upon linear mixing. For consistent results, gradient
pouring and polymerization are carried out under controlled conditions. The covalent
nature of the chemical bonds formed during the polymerization step results in a permanent
stability of the pH gradient within the matrix. Conflicting requirements during
the focusing procedure prevent any effective use of IPGs into capillary tubes (3): the
need to buffer with carrier ampholytes (CAs) the pH extremes caused by the migration
of the polymerization catalysts is contrasted by the adverse effects of the electroendosmotic
flow brought about by the addition of CAs to the gel phase. The demand for
the IPG gels to be backed by a binding support—they are usually cast on GelBond
foils—results in dimensional stability between 1-D and 2-D as a further assistance to
reproducibility.
Toward the aim of reproducibility, batch production and quality control as allowed by an
industrial process give the commercially available IPG strips (Immobiline DryStrip from
Pharmacia, Uppsala, Sweden) obvious advantages over homemade slabs. Moreover, the
pH course of the commercial product is being carefully characterized in chemicophysical
terms, by assessing the dissociation constants of the acrylamido buffers under the experimental
conditions relevant to the 1-D run of an IPG-DALT (4). The aim of this effort is to
connect focusing position reliably with pI (5); any discrepancy between computed and
experimental values for known proteins would then hint at the occurrence of posttranslational
modifications (6).
From the above it seems this chapter could shrink to the statement: Use the readymade
IPG strips according to the manufacturer’s instructions. However, there are reasons,
and not only of economical order, for laboratories to cast their own slabs. The
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
169