A fluorescence assay for monitoring and analyzing fusion of biological membrane vesicles in vitro

FEBS Letters

Stutzin, Andres¹ 

[1] Laboratory of Cell Biology and Genetics, NIADDK, NIH, Building 4, Room 312, Bethesda, MD 20892, USA

关键词: Membrane fusion Chromaffin granule Fluorescein Exocytosis Synexin;    PMSF;    phenylmethylsulfonylfluoride;    DTT;    dithiothreitol;   

DOI  :  10.1016/0014-5793(86)80341-6

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A new technique has been developed to study fusion of biological membrane vesicles. Bovine chromaffin granule ghosts (CGG) were loaded with fluorescein isothiocyanate-dextran (FITC-dextran) at self-quenching concentrations. Loaded ghosts were then made to fuse with empty CGG. Fusion was induced by synexin, a protein previously proposed to be involved in exocytosis. The fusion process was monitored by measuring the dequenching of the fluorescence. Dequenching occurred as FITC-dextran was diluted into the increased volume due to fusion with empty ghosts. Spurious signals from leakage or breakage of vesicles were removed by including a specific anti-fluorescein antibody in the reaction medium. This new technique may prove to be of more general use for studying membrane fusion processes in other systems.

【 授权许可】

Unknown