Casting Immobilized pH Gradients (IPGs)
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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.
Fig. 1. Structure of the IPG matrix. Different regions of an IPG made up with a basic and an acidic Immobiline are schematically drawn from cathode to anode. In each region, pH depends from the concentration ratio between the basic and the acidic dissociating groups. From the Henderson-Hasselbalch equation, where the concentration of the basic monomer is twice the concentration of the acidic monomer, pH equals the pK of the base, where the concentration of the basic monomer is one-half the concentration of the acidic monomer pH equals pK of the acid, and where the two concentrations are alike, pH equals the mean between the two pKs.