Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies

Abstract

A nitrocellulose gel transfer technique has been adapted to study the insulin-like growth factor (IGF) binding proteins of human serum. Normal and hypopituitary sera were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose or nylon membrane. Nonidet-P40 (3%) and Tween 20 (0.1%) were required for quenching and to allow detection of the IGF binding proteins by autoradiography after overlay with either 125I-labeled IGF I or IGF II. Several forms of IGF binding protein have been identified with molecular weights of 41,500, 38,500, 34,000, 30,000, and 24,000. Titration and competitive binding studies with IGF were performed on the transferred IGF binding proteins, indicating that binding proteins isolated by this technique can be characterized.