一种用于鉴定和表征可溶性核迁移因子的新型原位检测方法。

A novel in situ assay for the identification and characterization of soluble nuclear mobility factors.

作者信息

Elbi Cem, Walker Dawn A, Lewis Marcia, Romero Guillermo, Sullivan William P, Toft David O, Hager Gordon L, DeFranco Donald B

机构信息

Laboratory of Receptor Biology and Gene Expression, Building 41, Room B602, National Cancer Institute, Bethesda, MD 20892-5055, USA.

出版信息

Sci STKE. 2004 Jun 22;2004(238):pl10. doi: 10.1126/stke.2382004pl10.

DOI:10.1126/stke.2382004pl10

PMID:15213337

Abstract

The development of green fluorescent protein (GFP) technology combined with live cell microscopy techniques have revealed the dynamic properties of GFP-tagged proteins in the nucleus. The mobility of a GFP-tagged protein can be assessed using a quantitative photobleaching technique, fluorescence recovery after photobleaching (FRAP) analysis. FRAP experiments demonstrate that many nuclear proteins are highly mobile within the nucleus. However, the factors within the nucleus that regulate this mobility are not known. This is partly due to an absence of protocols that can be used to identify such nuclear mobility factors. We developed a novel in situ assay that combines a biochemical permeabilization and extraction procedure with a quantitative FRAP technique, a method we used to uncover a new functional role for molecular chaperones in the nuclear mobility of steroid receptors. This assay can readily be adapted to identify and characterize other nuclear mobility factors.

摘要

绿色荧光蛋白（GFP）技术与活细胞显微镜技术的结合，揭示了细胞核中GFP标记蛋白的动态特性。可以使用定量光漂白技术，即光漂白后荧光恢复（FRAP）分析，来评估GFP标记蛋白的流动性。FRAP实验表明，许多核蛋白在细胞核内具有高度的流动性。然而，细胞核内调节这种流动性的因素尚不清楚。部分原因是缺乏可用于鉴定此类核流动性因子的方案。我们开发了一种新的原位检测方法，该方法将生化通透和提取程序与定量FRAP技术相结合，我们用这种方法揭示了分子伴侣在类固醇受体核流动性中的新功能作用。这种检测方法可以很容易地用于鉴定和表征其他核流动性因子。