Pseudo‐ligand affinity chromatography and high‐voltage isoelectric focusing as a multiparameter method for separation and identification of plasma proteins

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Abstract
A system is described that utilizes physical‐chemical characteristics of macromolecules, not restricted to charge‐size variations, for the separation of the plasma proteins. The first step, using pseudo‐ligand affinity chromatography, separates the plasma proteins into fractions which were characterized by fused rocket immuno‐electrophoresis. Then electrophoresis in the form of high voltage gradient isoelectric focusing on ultrathin‐layer polyacrylamide gels is employed to further separate each of the fractions by charge differences. The advantages of this technique are that the proteins are maintained without noticeable denaturation, allowing a variety of direct functional tests. One is not restricted to only charge‐size variations for separation and the technique is a preparative‐to‐analytical procedure. Furthermore, the higher voltage gradients employed increase resolution of proteins significantly and require separation times of only 30–35 min. The limit of detectability is 50 μg/dl of protein, using silver diamine staining. The feasibility of this and related techniques as applied to the study of genetic polymorphisms and as an aid in the correlation and identification of spots obtained in dissociating‐denaturing two‐dimensional electrophoresis methods is emphasized.