Immunodetection of insulin after transfer from gels to nitrocellulose filters. A method of analysis in tissue extracts

Abstract

This report describes the development of a rapid method for detection of nanogram quantities of insulin in tissue extracts after electrophoresis. Following electrophoresis the proteins are transferred to nitrocellulose filters and treated with a photoreactive crosslinking agent. Filter bound insulin is detected by antiinsulin antibody and 125I-protein A, followed by autoradiography. The photoaffinity crosslinking is simple, rapid, and stable, and does not require reactive binding sites on derivatized paper. Under these conditions insulin maintains its immunoreactivity yet can be washed extensively to reduce nonspecific background; as little as 10 ng can be visualized. The method has proven to be useful for rapid analysis of qualitative as well quantitative differences in immunoreactive insulin in tissue extracts.