Kakita K, O'Connell K, Permutt M A
Diabetes. 1982 Jul;31(7):648-52. doi: 10.2337/diab.31.7.648.
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 nitro-cellulose 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 as quantitative differences in immunoreactive insulin in tissue extracts.
本报告描述了一种用于检测电泳后组织提取物中纳克量胰岛素的快速方法。电泳后，蛋白质被转移到硝酸纤维素滤膜上，并用光反应性交联剂处理。通过抗胰岛素抗体和125I-蛋白A检测滤膜结合的胰岛素，随后进行放射自显影。光亲和交联简单、快速且稳定，不需要衍生化纸上的反应性结合位点。在这些条件下，胰岛素保持其免疫反应性，但可以大量洗涤以减少非特异性背景；低至10纳克的胰岛素都可以可视化。该方法已被证明可用于快速分析组织提取物中免疫反应性胰岛素的定性和定量差异。
