Separation and Isolation of H and L Chains of IgG

Abstract

Reductive cleavage of the -S-S- bonds in an IgG molecule will liberate the H and L chains. If this cleavage is carried out near neutral pH and with mild reducing agents such as dithio-threitol, no further degradation and denaturation of the split products will occur. To block the sulfhydryl groups set free by the reductive cleavage, alkylation with iodoacetamide has to be carried out. The H and L chains can be separated by gel electrophoresis in 8 M urea-formic acid buffer, or by column chromatography on Sephadex G 100 equilibrated with 8 M urea in pH 6 Triscitrate buffer.