Characterization of insulin-like growth factor binding proteins by two-dimensional polyacrylamide gel electrophoresis and ligand blot analysis

Abstract

Insulin-like growth factor binding proteins (IGFBPs) in pregnant babooon serum and tissue culture media obtained following explant culture of uteri from pregnant baboons were characterized by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) followed by Western ligand blot analysis using 125I-labeled IGF-I. IGFBP-1 ( M r 30,000; p I 4–4.2), IGFBP-2 ( M r 34,000, p I 5.7–6.2), IGFBP-3 (doublet M r 42–48,000; pI 6.2–6.8), and IGFBP-4 ( M r 24,000; p I 5.7–6.0) were clearly separated from one another. The authenticity of IGFBP-1, -2, and -3 was verified by immunoprecipitation using polyclonal antibodies followed by ligand blotting. Specificity of 125I-labeled IGF-I binding to IGFBPs was also determined by competitive binding studies using unlabeled IGF-I and -II. This technique allows for the identification of IGFBPs in complex biological fluids on the basis of their characteristic M r and p I with or without the availability of specific antibodies and can be done rapidly using the mini 2D SDS-PAGE systems.