Immunofixation after electrofocusing: improved method for specific detection of serum proteins with determination of isoelectric points. I. Immunofixation print technique for detection of alpha-1-protease inhibitor

Abstract

An improved method is described for direct localization of human serum proteins in polyacrylamide gel with simultaneous determination of their isoelectric points (pI). The technique employs isoelectric focusing in thin-layer polyacrylamide gels to separate the serum proteins and the pH gradient is read at 4 degrees C with a dual-membrane surface microelectrode. Subsequently, the desired proteins are localized by immunofixation in the gel or by immunofixation-printing onto cellulose acetate strips soaked in specific antiserum. No sectioning of the electrofocused gel is necessary, and the entire technique can be completed in less than 14 h. When this method is applied to the detection of the genetic variants of alpha-1-antitrypsin (alpha-1-protease inhibitor) (A1Pi system), the results indicate that it can be used to specifically localize serum proteins whose pI's differ by as little as 0.01 pH units. The resolution afforded is especially evident in the analysis of A1Pi M variants.