通过蛋白质免疫印迹分析对相关蛋白质进行定量。

Quantitation of related proteins by Western blot analysis.

作者信息

Uhl J, Newton R C

机构信息

E.I. du Pont de Nemours and Co., Medical Products Department, Glenolden, PA 19036.

出版信息

J Immunol Methods. 1988 May 25;110(1):79-84. doi: 10.1016/0022-1759(88)90085-3.

DOI:10.1016/0022-1759(88)90085-3

PMID:3286776

Abstract

A spectrophotometric assay for quantitation of related proteins following Western blot analysis is described. Proteins were separated by polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. In the system utilized in this study, immobilized interleukin-1 (IL-1) proteins were identified by ELISA reaction using a rabbit antibody specific for IL-1 beta. Subsequently, the membrane was probed with goat anti-rabbit antibody conjugated to horseradish peroxidase and IL-1 proteins were detected by incubation with 4-chloro-1-naphthol and hydrogen peroxide. The stained IL-1 bands were cut out, exposed to the peroxidase chromogenic substrate, o-phenylenediamine (OPD), and hydrogen peroxide and the rate of reaction was determined spectrophotometrically at 490 nM. A linear relationship between enzyme activity and IL-1 concentration was observed from 10 to 1000 ng/lane. Thus, this represents a sensitive and specific method for quantitating small amounts of IL-1 in complex protein mixtures.

摘要

本文描述了一种用于蛋白质印迹分析后相关蛋白质定量的分光光度测定法。蛋白质通过聚丙烯酰胺凝胶电泳分离，并通过电泳转移至硝酸纤维素膜上。在本研究使用的系统中，使用针对白细胞介素-1β（IL-1β）的兔抗体，通过酶联免疫吸附测定（ELISA）反应鉴定固定化的IL-1蛋白质。随后，用与辣根过氧化物酶偶联的山羊抗兔抗体探测膜，并通过与4-氯-1-萘酚和过氧化氢孵育来检测IL-1蛋白质。将染色的IL-1条带切下，暴露于过氧化物酶显色底物邻苯二胺（OPD）和过氧化氢中，并在490 nM处通过分光光度法测定反应速率。在每泳道10至1000 ng范围内观察到酶活性与IL-1浓度之间呈线性关系。因此，这是一种用于定量复杂蛋白质混合物中少量IL-1的灵敏且特异的方法。